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Antibody Applications

Method Booklet 2

Table of Contents

Introduction................................................2

ELISA ..............................................................4

Formulating the assay ................................4

Method ......................................................7

Flow Cytometry........................................14

Extracellular Staining ................................14

Intracellular Staining ................................31

Immunohistochemistry ........................37

Immunoprecipitation/Western ..........41Stimulation ..............................................43Cell Extracts..............................................44Protein Assay ............................................46Immunoprecipitation ................................47Western Blotting ......................................51

Neutralization ..........................................63

Appendix ....................................................64

Antibody-Peptide Competition..................64

ELISA Troubleshooting Guide ..................65

Guidelines for the Development of

Western blotting method ......................69

Suggested Vendors....................................71

References ..................................................72

Antibody Applications

Neutralization is another technique described to study the interaction between a ligandand its receptor. BioSource International offers a complete line of neutralizing antibod-ies for human, mouse, rat and swine cytokines for this purpose.

We welcome your feedback on the methods described. Please contact us with your com-ments.

Disclaimer

The procedures presented in this manual are accurate to the best of our knowledge.BioSource International, Inc. makes no warranties either express or implied as to any mat-ter whatsoever, including, without limitations, the condition of the products, their mer-chantability, or fitness for any particular use. BioSource International, Inc, shall not beheld liable for any direct, indirect, incidental, or consequential damages, including with-out limitation, loss of profit, loss of business, or other loss which may be based directlyor indirectly upon the sale, use of products, or inadequacy of product for any purpose orby any defect or deficiency therein, even if BioSource International, Inc., knew or shouldhave known of the possibility of such loss.

The products listed in this booklet are for research use only, andare not intended for use in diagnostic procedures.

IntroductionThe goal of this manual is to provide users with easy to follow written methods withwhich to obtain meaningful results. The research and development staff of BioSourceInternational, Inc. has documented their protocols for various methods and detailed theclues which will aid in successful testing of our antibody products. These details includebuffer formulations and specific recipes for preparing cells and tissues. Specific data gen-erated at BioSource International is given to illustrate typical results when following theseprotocols. Suggested product part numbers are also given to guide you in your selectionof appropriate reagents. As always, your feedback has contributed to the initiative for thispublication and continues to effect improvements in the materials which are suppliedwith our products. Please contact our Technical Service Staff with any inquiries regard-ing this manual

The first method which is described in this publication is the Enzyme-Linked-ImmunoSorbent Assay (ELISA). This method is a benchmark for quantitation of patholog-ical antigens and there are indeed many variations to this method. ELISAs are adaptableto high-throughput screening because results are rapid, consistent and relatively easy toanalyze. We have obtained the best results when utilizing a Sandwich format, whichcombines pre-matched capture antibody, Biotin-labeled detection antibody and our pro-prietary Horseradish Peroxidase conjugated Streptavidin. The resulting signal providesdata which is very sensitive and highly specific. BioSource International offers a completeline of antibodies for the quantitation of human, mouse, rat and swine cytokines.Additionally, a complete line of ready-to-use ELISA kits are available for the measurementof human, rat, mouse, monkey and swine cytokines, chemokines, growth factors andadhesion molecules.

Following the ELISA section are instructions for staining cells (Extracellular andIntracellular) for detection by flow cytometry. FACS analysis is a method that was devel-oped to identify, purify and characterize lymphocytes. This method has allowed thedevelopment of antibodies which are now used in the diagnosis of various leukemias andother blood cell-based disorders. BioSource International offers a choice of completekits, antibodies and reagents for the preparation of blood samples for extra- and intracel-lular staining of human, rat and mouse cytokines and their receptors.

Antibodies can also be used to identify the anatomic distribution of antigens within tis-sues and cells. For immunohistochemistry applications, Biosource International offersfluorescent-labeled antibodies as well as enzyme-linked antibodies for the staining offixed cells and tissues by microscopy.

Immunoprecipitation (IP) and Western blotting methods are described for the isolationand analysis of phosphorylated proteins. Detailed methods are given for the stimulationof phosphoproteins and the preparation of cell extracts for IP. Alternatives are given forcolorimetric as well as enhanced chemiluminescent detection. A complete line of affin-ity-purified phosphorylation-state antibodies for cell signalling are now available fromBioSource.

ELISAIntroduction

A typical Sandwich ELISA format involves a specific capture antibody, samples, abiotinylated detection antibody, a Streptavidin-HRP (SAV) conjugate, chromogen andstop solution. Antigen will bind to the immoblilized capture antibody and to thebiotinylated detection antibody. The SAV binds to the detection antibody to completethe Sandwich. A substrate solution is added, acted upon by the enzyme conjugatedSAV, and effects a color change. The intensity of this color change is proportional to theamount of antigen in the original sample. Several important elements of constructing aworking assay are briefly discussed, followed by a detailed protocol. Lastly, a completelisting of pre-matched reagents is given (see page 11).

Formulating the assayAntibodies

Certainly one of the most important characteristics of a good assay is the selection of agood antibody pair and a suitable antigen. Once they have been selected, the appro-priate titers and volumes must be chosen. A standard curve range of 0-2 ng/mL is gen-erally acceptable for measurement of cytokines in most sample types. Both the captureand detecting antibodies must be independently titered and working volumes chosen.A titration scheme using the standard as the indicator is given in Figure 1.

1. The capture antibody can be tested in concentrations ranging from 0.5-10 µg/mL.Serial dilutions can be prepared starting with 10 µg/mL and the coating volumeshould be 100 µL-200 µL.

2. The biotinylated detection antibody can be tested in concentrations ranging from0.05-2.0 µg/mL. The volume of detection antibody can be 50 µL to 100 µL.

Figure 1: Titration of Antibodies

The best working conditions will satisfy the following criteria:

Incubations: Time and Temperature

Incubation times and temperatures for all steps should be empirically determined. In gen-eral, the temperature of incubation will contribute to determination of the incubationtime. For example, if the standard incubation is performed at 4°C, then usually anovernight incubation will be required to obtain a desirable signal. When incubations areperformed at 37°C, then much shorter incubation times are necessary.

1. Coating of the microwells may be performed at room temperature or at 4°C.Optimal saturation of the plastic should occur over 4-48 hours. Note, however, thatvarious plastics must also be tested for optimal performance.

2. The temperature of the detection antibody incubation can also be varied from 4-37°C. The incubation time is dependent on sample recovery and desired signal forboth a separate and combined assay. Generally 30 minutes-2 hours is enough timefor the detection antibody incubation step.

Signal Intensity

With the protocols given, the non-specific signal (zero value) should be less than 0.2 O.D.units. If higher O.D.s are observed, this is not acceptable. To lower the non-specific sig-nal:

1. Decrease the incubation times or volumes of either the sample and/or the biotiny-lated detection antibody.

2. Improve the washing procedure. Use the wash buffer recommended in the packageinsert. When washing, fill the wells by delivering a forceful stream of buffer into thewells. Allow the wells to soak for 15-30 seconds. Aspirate the wash buffer com-pletely. Repeat 2-5 times. Following the final aspiration, tap the plate forcefully onabsorbent paper until all of the residual buffer is removed. Proceed immediately tothe next step. Omitting Tween-20 from the wash buffer has also been observed toreduce the non-specific signal.

3. Increase the incubation and concentration of the blocking solution. The use ofcasein as a blocking agent may reduce non-specific signal.

4. Optimize the Standard Diluent/Assay Buffer by decreasing or increasing the amountof animal serum or BSA. (Refer to Buffers section.)

-standard 0: < 0.2 O.D.-standard 3: 1.5-2.5 O.D.-standard 1standard 0

: ≥ 2

1 2 3 4 5 6 7 8 9 10 11 12

A STD 0

B Coating STD 1

C Concentration 1 Concentration 2 STD 2

D STD 3

E STD 0

F Coating Coating STD 1

G Concentration 3 Concentration 4 STD 2 H STD 3

1 2 3 1 2 3Biotin Concentration

Coa ngti

ELISA: Method

The following protocol has been developed using antibody pairs chosen for their speci-ficity to the target antigen, reproducibility of results, and precision in quantitation. Themajority of antibodies offered are monoclonal antibodies which consistently recognize asingle epitope on the target. BioSource International offers monoclonals rather than poly-clonal antisera because these qualities reduce the number of variables an investigator willhave to consider when preparing a home-brew ELISA. Regardless of the manufacturer,the antibody should have a high purity (at least >95%) and preferably have no addedserum proteins.

Purpose: To quantitate a specific protein against a known quantity of standard proteinusing specific antibodies.

Materials and EquipmentCoating antibody Plate washing deviceDetection antibody (Biotin) Plate coversDiluents Microwell plate readerStrepdavidin-HRP Microwells (96 per plate)Chromogen (TMB) Refrigerator (4°C)Stop solution Incubator (37°C)Wash Buffer Pipettors

Note: All buffers are shown in bold type, formulations are given on page 9, Table 1.

Plate Coating1. Dilute Coating Antibody in Coating Buffer to the concentration recommended on

the accompanying antibody information sheet. Refer to vial label for concentrationof Coating Antibody. Avoid polystyrene containers when storing or diluting anti-body. If the standard curve signals are not as high as desired, higher coating con-centrations of antibody can be tried.

2. Add 100 µL of diluted Coating Antibody per well to polystyrene microplates. (e.g.,Dynex Immulon 2 HB or Nunc Maxisorp). Other polystyrene plates may also besuitable.

3. Cover the plates and incubate overnight (12 to 18 hours) at 2 - 8°C. Incubating forlonger periods of time, such as over the weekend, should not cause any problems.

4. Aspirate the coating antibody from the wells and tap on absorbent paper to removeexcess liquid.

5. Add 400 µL per well of Wash Buffer, incubate for 15-30 seconds, and aspirate.6. Add 300 µL of Blocking Solution to each well. Cover the plates and incubate for

1 to 2 hours at room temperature. Excessive blocking should be avoided. If back-grounds are higher than desired, try substituting either casein or calf serum in placeof the BSA found in the Blocking Solution.

Buffers

1. The coating buffer most commonly used is carbonate buffer (Coating Buffer B).Phosphate buffers have also worked well in our labs. The ionic strength (200-400 nM)and pH (3-10 pH units) of these buffers can be altered to achieve more efficient cap-ture antibody binding.

2. Assay diluent buffers usually contain a serum protein which helps to mimic the pro-teins contained in sample types. In most ELISA systems, bovine serum albumin isthe protein recommended. In some cases where recovery is poor (e.g., less than80%), increasing the concentration of BSA or addition of animal serum will increaserecovery.

3. Streptavidin diluents affect not only the stability of the enzyme, but also the rate ofthe color change. Horseradish Peroxidase, the enzyme recommended here, is activeover a range of pH, but if the pH is too low, the HRP may become unstable.Detergent, temperature and molarity of the buffer are all variables which, whenaltered, can affect the performance of the HRP.

Sensitivity

Sensitivity or Minimum Detectable Concentration (MDC) is confirmed by assaying a min-imum of 20 replicates of the zero standard in a single assay. The concentration extrapo-lated from the standard curve of the average ODs of the standard 0 replicates + 2 SD isthe MDC. This value represents the lowest value read from the standard curve that can bestatistically differentiated from zero.

Assay sensitivity can be estimated from the example standard curve provided on the infor-mation sheet. If the specific procedure is closely followed, the example standard curvecan be duplicated.

The desired level of sensitivity and range of the curve may affect the concentrations ofreagents used. For example, if the range of the curve is narrow, a higher concentration ofstreptavidin may be required. The optical limitations of the plate reader must also be con-sidered.

Assay precision and non-specific binding affect the assay sensitivity. Precision can beimproved with the use of calibrated pipettes, careful technique and vigorous washing.Choose assay conditions which show the highest signal to noise ratio.

14. Stop the reaction by addition of 50 or 100 µL of Stop Solution to each well accord-ing to the manufacturer’s instructions.

15. Read microplate at 450 nm within 30 minutes of adding Stop Solution. Prior tostopping color development, plate can be read at 650 nm to determine if desiredOD’s have been obtained.

16. Calculate the average optical density at 450 nm for all Standards, Controls andSamples. Construct a standard curve by plotting each Standard optical density(ordinate) vs. the Standard concentration (abscissa) on semi-log graph paper. Forplate readers with automated standard curve calculation capability, a log-log or fourparameter curve fit algorithm may provide the best curve fit.

17. Determine the concentration of each unknown sample from the standard curve.

Please see the troubleshooting guide in the appendix for additional tips.

Table 1:

• This procedure contains only recommendations. Investigators are advised to determine optimal buffer formulations, concentrations and incubation times for individual application.

7. Aspirate the Blocking Solution from the wells and tap on absorbent paper toremove excess liquid. Plates can now be used. Alternatively, if the plates are not tobe used immediately allow to dry overnight at room temperature, then store at 2 -8°C in a plastic bag, with desiccant, for up to a week.

8. Prior to starting the assay, wash the microplate 3 to 6X with 400 µL per well of WashBuffer and tap on absorbent paper to remove all excess liquid after the final wash.At this point do not allow wells to dry completely at any time.

ELISA Method1. Add standards and samples to the coated microplate.2. Dilute standards and samples in Standard Diluent/Assay Buffer or in the assay

matrix most relevant to your samples (e.g., cell culture medium containing 10%fetal calf serum).

3. Add 100 µL of standards, samples and controls to appropriate wells in duplicate.Controls can include a reagent blank (zero standard control) and a substrate blank.Incubate now, for the time determined during assay optimization. As a startingpoint, use one hour. If a co-incubation is desired, continue to Step 4.

Note: Some assays may require separate incubation of standards, samples and controlsprior to addition of the biotinylated detection antibody (Step #4). Refer to the accompa-nying antibody information sheet for specific instructions under standard incubation rec-ommendations.

4. Dilute biotinylated Detection Antibody in Standard Diluent/Assay Buffer to theconcentration recommended on the accompanying antibody information sheet.

5. Add 50 or 100 µL of diluted Detection Antibody to each well except chromogenblank, cover the plate and incubate for the time and temperature determined dur-ing the optimization of the assay. For a starting point, use one hour. If you are per-forming a co-incubation (sample and Biotin antibody) use two hours.

6. Aspirate solution from wells.7. Wash the microplate 3 to 6X with 400 µL per well of Wash Solution and tap on

absorbent paper to remove all excess liquid after the final wash.8. Dilute Streptavidin-HRP conjugate according to the manufacturer’s instructions.

Streptavidin-HRP may be diluted in Standard Diluent/Assay Buffer if not otherwisespecified by the package insert.

9. Add 100 µL of diluted Streptavidin-HRP per well, cover the microplate and incubateat room temperature for 15 to 45 minutes. (Note: Dilution and incubation time willvary depending on the lot).

10. Aspirate solution from wells.11. Wash the microplate 3 to 6X with 400 µL per well of Wash Solution and tap on

absorbent paper to remove all excess liquid after the final wash.12. Prepare TMB (tetramethylbenzidine) substrate according to the manufacturer’s

instructions.13. Add 100 µL of TMB to each well and incubate in the dark at room temperature for

10 to 60 minutes (generally 30 minutes) according to the manufacturer’s instruc-tions. (Note: Incubation time will vary depending on manufacturer and lot).

Solution FormulationCoating Buffer A 8.0 g NaCl

1.42 g Na2HPO4H2O0.2 g KH2PO4

0.2 g KClq.s. to 1 liter with distilled H2O, pH 7.4

Coating Buffer B 4.3 g NaHCO35.3 g Na2CO3q.s. to 1 liter with distilled H2O, pH 9.4

Blocking Solution 8.0 g NaCl1.42 g Na2HPO4

.2H2O0.2 g KH2PO4

0.2 g KCl5.0 g bovine serum albumin (fraction V)q.s. to 1 liter with distilled H2O, pH 7.4

Standard Diluent/Assay Buffer 8.0 g NaCl1.42 g Na2HPO4

.2H2O0.2 g KH2PO40.2 g KCl5.0 g bovine serum albumin (fraction V)1 mL Tween 20q.s. to 1 liter with distilled H2O, pH 7.4

Wash Buffer 9.0 g NaCl1 mL Tween 20q.s. to 1 liter with distilled H2O, pH 7.4

Stop Solution 1.8 N H2SO4

Figure 2: This diagram illustrates a typical sandwich ELISA. ELISA Antibody SetsThe following products are paired antibodies with the matching cytokine control.

Human ELISA ProductsCytokine Product Part Number SizeEGF Capture antibody AHG0064 0.5 mg

Detection antibody AHG9069 0.1 mgRecombinant standard SD088 1 vial

EMAP-II Capture antibody AHC9584 0.5 mgDetection antibody AHC9589 0.1 mgRecombinant standard SD095 1 vial

Eotaxin Capture antibody AHC6054 0.5 mgDetection antibody AHC6069 0.1 mgRecombinant standard SD073 1 vial

Fas Capture antibody AHS9544 0.5 mgDetection antibody AHS9569 0.1 mgRecombinant standard SD053 1 vial

G-CSF Capture antibody AHC2034 0.5 mgDetection antibody AHC2939 0.1 mgRecombinant standard SD091 1 vial

GM-CSF Capture antibody AHC2812 0.5 mgDetection antibody AHC2919 0.1 mgRecombinant standard SD912 1 vial

ICAM-1 Capture antibody AHS5444 0.5 mgDetection antibody AHS5429 0.1 mgRecombinant standard SD051 1 vial

IFN-γ Capture antibody AHC4432 0.5 mgDetection antibody AHC4539 0.1 mgRecombinant standard SD911 1 vial

IL-1α Capture antibody AHC0912 0.5 mgDetection antibody AHC0419 0.1 mgRecombinant standard SD901 1 vial

IL-1β Capture antibody AHC0612 0.5 mgDetection antibody AHC0519 0.1 mgRecombinant standard SD902 1 vial

IL-1ra Capture antibody AHC0112 0.5 mgDetection antibody AHC0219 0.1 mgRecombinant standard SD916 1 vial

IL-3 Capture antibody AHC0832 0.5 mgDetection antibody AHC0939 0.1 mgRecombinant standard SD904 1 vial


IL-5 Capture antibody AHC0954 0.5 mgDetection antibody AHC0859 0.1 mgRecombinant standard SD013-2 1 vial




IL-10 Capture antibody AHC8102 0.5 mgDetection antibody AHC7109 0.1 mg

Streptavidin ProductsDescription Part Number SizeStreptavidin SNN1001 1.0 mgStreptavidin Alk-Phos SNN1005 1.0 mgStreptavidin FITC SNN1008 1.0 mgStreptavidin HRP SNN1004 1.0 mgStreptavidin HRP ELISA Grade SNN2004 1.0 mgStreptavidin R-PE SNN1007 0.5 mg

Swine ELISA ProductsCytokine Product Part Number SizeIFN-γ Capture antibody ASC4934 0.5 mg

Detection antibody ASC4839 0.1 mgRecombinant standard SD066 1 vial

IL-2 Capture antibody ASC0924 0.5 mgDetection antibody ASC0829 0.1 mgRecombinant standard SD085 1 vial



Rat ELISA ProductsCytokine Product Part Number SizeIL-2 Capture antibody ARC0924 0.5 mg

Detection antibody ARC0829 0.1 mgRecombinant standard SD052 1 vial

IL-4 Capture antibody ARC0944 0.5 mgDetection antibody ARC0749 0.1 mgRecombinant standard SD049 1 vial

IL-10 Capture antibody ARC9104 0.5 mgDetection antibody ARC7109 0.1 mgRecombinant standard SD048 1 vial

MIP-2 Capture antibody ARC1074 0.5 mgDetection antibody ARC1079 0.1 mgRecombinant standard SD038 1 vial

Mouse ELISA Products (contined)Cytokine Product Part Number SizeIL-4 Capture antibody AMC0044 0.5 mg

Detection antibody AMC0949 0.1 mgRecombinant standard SD029-2 1 vial

IL-5 Capture antibody AMC0052 0.5 mgDetection antibody AMC0959 0.1 mgRecombinant standard SD076 1 vial

IL-6 Capture antibody AMC0864 0.5 mgDetection antibody AMC0969 0.1 mgRecombinant standard SD030-2 1 vial

IL-10 Capture antibody AMC8104 0.5 mgDetection antibody AMC0109 0.1 mgRecombinant standard SD031-2 1 vial


TNF-α Capture antibody AMC3014 0.5 mgDetection antibody AMC3719 0.1 mgRecombinant standard SD023-2 1 vial

Human ELISA ProductsCytokine Product Part Number SizeIL-12 Capture antibody AHC8122 0.5 mg

Detection antibody AHC7129 0.1 mgRecombinant standard SD910 1 vial




Leptin Capture antibody AHC6094 0.5 mgDetection antibody AHC6109 0.1 mgRecombinant standard SD092 1 vial

MIP-1α Capture antibody AHC6034 0.5 mgDetection antibody AHC6049 0.1 mgRecombinant standard SD063 1 vial

MIP-1β Capture antibody AHC6114 0.5 mgDetection antibody AHC6129 0.1 mgRecombinant standard SD094 1 vial

MIP-5 Capture antibody AHC6074 0.5 mgDetection antibody AHC6089 0.1 mgRecombinant standard SD087 1 vial

RANTES Capture antibody AHC6014 0.5 mgDetection antibody AHC6029 0.1 mgRecombinant standard SD037 1 vial

TIMP-1 Capture antibody AHC1494 0.5 mgDetection antibody AHC9499 0.1 mgRecombinant standard SD074 1 vial

TNF-α Capture antibody AHC3712 0.5 mgDetection antibody AHC3419 0.1 mgRecombinant standard SD913 1 vial

TNF-RI Capture antibody AHR3912 0.5 mgDetection antibody AHR3819 0.1 mgRecombinant standard SD914 1 vial

TNF-RII Capture antibody AHR3022 0.5 mgDetection antibody AHR3929 0.1 mgRecombinant standard SD915 1 vial

VCAM-1 Capture antibody AHT0624 0.5 mgDetection antibody AHT0619 0.1 mgRecombinant standard SD050 1 vial

Mouse ELISA ProductsCytokine Product Part Number SizeGM-CSF Capture antibody AMC2014 0.5 mg

Detection antibody AMC2929 0.1 mgRecombinant standard SD075 1 vial

IFN-γ Capture antibody AMC4934 0.5 mgDetection antibody AMC4739 0.1 mgRecombinant standard SD024-2 1 vial

IL-1β Capture antibody AMC0914 0.5 mgDetection antibody AMC0719 0.1 mgRecombinant standard SD026-2 1 vial


Flow Cytometry

A. Extracellular Staining

IntroductionExtracellular Staining of monoclonal antibodies has been used extensively to character-ize cell surface antigens over the past 20 years. Flow cytometry data have been used toidentify the Clusters of Differentiation (CD) and to group antibodies based on their recog-nition of these antigens. Formal leukocyte workshops were formed to identify the similarstaining patterns of grouped antibodies not only for human antigens, but for other speciesas well. The use of flow cytometry for extracellular staining continues to provide usefuldata for identifying and separating cell populations. A complete listing of BSI CD prod-ucts are shown on pages 16-30.

Purpose: For staining proteins on the surface of the cell with directly conjugated anti-bodies for analysis by flow cytometry.

Materials and EquipmentSample cells or whole blood Ice12 x 75 mm test tubes Vortex mixerPBS/0.1% sodium azide/1% FBS Lysis Solution (whole blood)Fluorescent-labelled CD Antibody Flow cytometerCentrifuge PBS, 0.5% paraformaldehyde, 4°CPBS/NaN3, 4°C

Preparation of Cells

Note: If sample is whole blood, see next protocol.

1. Prepare the desired biological cells according to the appropriate protocol. Adjustthe concentration of the cells to 2 x 107 cells per mL (or 1 x 106 cells per 50 µL:each test consists of 1 x 106 cells), diluting with PBS/sodium azide/FBS, as neces-sary. The cells can be isolated up to 4 hours before being stained as long as theyare kept on ice.Note: In some cases, use of a non-specific binding or blocking agent may bedesired. If so, add the blocking agent and incubate at room temperature for 10minutes prior to the addition of antibody to the cells.

2. Dilute the fluorochrome conjugated antibody appropriately for the type of stainbeing done. Use PBS/sodium azide/FBS for the dilution. Typically, 0.125-0.5 µLof conjugated monoclonal antibody stains 106 cells. Dilute the antibody so that avolume of 10-20 µL are added to the cells.

3. Pipette 50 µL of the previously isolated biological cells (Step 1) into the bottom ofeach labeled 12 x 75 mm test tube. Add the appropriate volume of diluted anti-body and mix gently. Incubate the cells with the antibody at 2-8°C for 30 minutesin the dark. All conjugated antibodies (FITC, R-PE, Cy5/R-PE) for double or triplestaining can be added simultaneously at this point and do not require additionalincubations.

4. After incubation, remove the unbound antibody from the cells by washing with1.0 mL of PBS/sodium azide/FBS.4.1 Pipette the PBS/NaN3/FBS in to each tube, vortex, then centrifuge at 350 x g

for 5-7 minutes at 2-8°C. Carefully aspirate the supernatant leaving about 50-100 µL in the bottom of each tube.

4.2 Repeat this process for a total of 3 washes.5. If flow cytometric analysis is to be performed the same day, resuspend the cells in

0.5 mL of cold PBS/NaN3 after aspirating the supernatant. Vortex and analyze thecells. If analysis will not be performed the same day, the cells may be fixed in 0.5mL cold PBS with 0.5% paraformaldehyde and stored at 2-8°C in the dark inbuffer containing 0.1 NaN3 for as long as 48 hours.

Alternate Protocol for Staining Whole BloodIf whole blood is being used, several modifications can be made to the above protocol.Typically, 100 µL of freshly isolated whole blood is used for staining purposes.

1. Pipette the desired volume of conjugated antibodies into 12 x 75 mm tubes thenadd 100 µL whole blood into the tube, being careful not to pipette down the side.For double and triple staining, all conjugated antibodies can be added simultane-ously.

2. Incubate cells for 30 minutes at 4°C in the dark.3. The RBCs will need to be lysed so that they do not alter or interfere with the

FACScan analysis. To lyse cells, add 2.0 mL 1X lysing solution (Becton DickinsonLysis Solution) to whole blood.

4. Vortex to mix and leave cells for 2-5 minutes until solution begins to clear (thesolution will become a translucent light red color).

5. Spin cells at 350 x g for 5 minutes and aspirate supernatant then continue asdirected in Step 4.1 of the above protocol.

Indirect Staining of Extracellular ProteinsIf an unconjugated antibody is being used with double or triple staining then the primaryantibody should be incubated with the cells for 30 minutes at 4°C and the cells washed2 times prior to the addition of the conjugated secondary. The secondary antibody isincubated for 30 minutes at 4°C and removed by washing with PBS/NaN3/FBS. At thistime, add any conjugated antibodies and repeat staining protocol.

Notes: For any double or triple staining procedure where the cells are being analyzed byflow cytometry it is important that the instrument is properly calibrated and that the com-pensation of the lasers is set up prior to collecting data. For each flow cytometer, the com-pensation will be slightly different for a double or triple staining experiment and it is bestto consult manufacturer’s guidelines on how to set up your particular instrument.

Antibodies for Flow Cytometry-Extracellular Staining Product Description Part Number SizeHuman CD9 R-PE AHS0907 100 tests/1 mLHuman CD9 Unconj. AHS0921 0.1 mg/0.1 mLHuman CD10 Unconj. AHS1001 200 tests/2 mLHuman CD10 Azide-free AHS1002 0.1 mg/0.1 mLHuman CD10 FITC AHS1008 100 tests/1 mLHuman CD10 R-PE AHS1007 100 tests/1 mLHuman CD11a Azide-free AHS1112 0.1 mg/0.1 mLHuman CD11a FITC AHS1118 100 tests/1 mLHuman CD11a R-PE AHS1117 100 tests/1 mLHuman CD11a Unconj. AHS1161 0.1 mg/1 mLHuman CD11a Biotin AHS1169 100 tests/1 mLHuman CD11a FITC AHS1168 100 tests/1 mLHuman CD11b Unconj. AHS1121 0.1 mgHuman CD11b Biotin AHS1129 100 tests/1 mLHuman CD11b FITC AHS1128 100 tests/1 mLHuman CD11b Unconj. AHS1142 0.2 mg/1 mLHuman CD11b FITC AHS1148 100 tests/1 mLHuman CD11b R-PE AHS1147 100 tests/1 mLHuman CD11c Unconj. AHS1152 0.2 mg/1 mLHuman CD11c FITC AHS1158 100 tests/1 mLHuman CD11c R-PE AHS1157 100 tests/1 mLHuman CD11c AHS1131 100 µg/1 mLHuman CD13 Unconj. AHS1301 200 tests/2 mLHuman CD13 Azide-free AHS1302 100 µg/100 µLHuman CD13 FITC AHS1308 100 tests/1 mLHuman CD14 Azide-free AHS1412 100 µg/100 µLHuman CD14 Azide Free AHS1402 100 µgHuman CD14 FITC AHS1418 100 testsHuman CD14 R-PE AHS1417 100 testsHuman CD14 Unconj. AHS1421 100 µg/100 µLHuman CD15 Unconj. AHS1501 200 tests/2 mLHuman CD15 Azide-free AHS1502 100 µg/100 µLHuman CD16 Unconj. AHS1601 200 tests/2 mLHuman CD16 FITC AHS1608 100 tests/1 mLHuman CD16 R-PE AHS1607 100 tests/1 mLHuman CDw17 Unconj. AHS1701 200 tests/2 mLHuman CD18 Unconj. AHS1821 200 tests/2 mLHuman CD18 Azide-free AHS1822 100 µgHuman CD18 Biotin AHS1829 100 tests/1 mLHuman CD18 FITC AHS1828 100 tests/1 mLHuman CD18 Unconj. AHS1811 100 tests/1 mLHuman CD18 Biotin AHS1819 100 testsHuman CD18 FITC AHS1818 100 tests

Human Leukocyte CD AntigensProduct Description Part Number SizeHuman CD1a Azide-free AHS0181 200 testsHuman CD1a FITC AHS0188 100 testsHuman CD1a (LO-CD 1a) AHS0121 100 µg/100 µLHuman CD1b Unconj. AHS0161 200 tests/2 mLHuman CD1d Unconj. AHS0151 200 tests/2 mLHuman CD2 Unconj. AHS0212 0.1 mgHuman CD2 FITC AHS0208 100 tests/1 mLHuman CD2 Biotin AHS0219 100 tests/1 mLHuman CD2 Biotin AHS0209 100 tests/1 mLHuman CD3 Azide-free AHS0312 100 µg/1 mLHuman CD3 Biotin AHS0319 100 tests/1 mLHuman CD3 FITC AHS0318 100 tests/1 mLHuman CD3 R-PE AHS0317 100 tests/1 mLHuman CD4 Azide-free AHS0412 100 µg/100 µLHuman CD4 Biotin AHS0419 100 tests/1 mLHuman CD4 FITC AHS0418 100 tests/1 mLHuman CD4 R-PE AHS0417 100 tests/1 mLHuman CD5 Unconj. AHS0501 200 tests/2 mLHuman CD5 Azide-free AHS0502 100 µg/100 µLHuman CD5 FITC AHS0508 100 tests/1 mLHuman CD5 R-PE AHS0507 100 tests/1 mLHuman CD5 Unconj. AHS0511 200 tests/2 mLHuman CD5 Azide-free AHS0512 100 µg/100 µLHuman CD5 FITC AHS0518 100 tests/1 mLHuman CD5 R-PE AHS0517 100 tests/1 mLHuman CD5 Unconj. AHS0521 100 µg/100 µLHuman CD6 Unconj. AHS0601 200 tests/2 mLHuman CD6 Azide-free AHS0602 100 µg/100 µLHuman CD6 FITC AHS0608 100 tests/1 mLHuman CD6 Unconj. AHS0621 100 µg/100 µLHuman CD7 Unconj. AHS0701 200 tests/2 mLHuman CD7 Azide-free AHS0702 100 µg/100 µLHuman CD7 FITC AHS0708 100 tests/1 mLHuman CD8 Azide-free AHS0812 100 µg/100 µLHuman CD8 Biotin AHS0819 100 tests/1 mLHuman CD8 FITC AHS0818 100 tests/1 mLHuman CD8 R-PE AHS0817 100 tests/1 mLHuman CD9 Unconj. AHS0901 200 tests/2 mLHuman CD9 FITC AHS0908 100 tests/1 mL

Product Description Part Number SizeHuman CD33 R-PE AHS3317 100 testsHuman CD34 Unconj. AHS3401 200 µg/2 mLHuman CD34 Biotin AHS3409 100 tests/1 mLHuman CD34 FITC AHS3408 100 tests/1 mLHuman CD34 R-PE AHS3407 100 tests/1 mLHuman CD35 Unconj. AHS3502 200 µgHuman CD35 FITC AHS3508 100 tests/1 mLHuman CD35 R-PE AHS3507 100 tests/1 mLHuman CD36 Unconj. AHS3602 200 µgHuman CD36 FITC AHS3608 100 tests/1 mLHuman CD36 AHS3612 100 µgHuman CD37 Unconj. AHS3701 200 µg/2 mLHuman CD37 FITC AHS3708 100 tests/1 mLHuman CD37 Unconj. AHS3791 200 µg/1 mLHuman CD39 Unconj. AHS3911 200 testsHuman CD40 Azide-free AHS4002 100 µg/100 µLHuman CD40 Biotin AHS4009 100 tests/1 mLHuman CD40 FITC AHS4008 100 tests/1 mLHuman CD40 R-PE AHS4007 100 tests/1 mLHuman CD41a Unconj. AHS4101 200 tests/2 mLHuman CD41a FITC AHS4108 100 tests/1 mLHuman CD41a R-PE AHS4107 100 tests/1 mLHuman CD41a R-PE AHS4111 100 µg/100 µLHuman CD42a Unconj. AHS4231 200 testsHuman CD42b FITC AHS4238 100 tests/1 mLHuman CD43 Unconj. AHS4311 200 µgHuman CD43 FITC AHS4318 100 testsHuman CD44 Unconj. AHS4401 200 tests/2 mLHuman CD44 FITC AHS4408 100 tests/1 mLHuman CD44 R-PE AHS4407 100 tests/1 mLHuman CD44std Unconj. AHS4411 100 µg/1 mLHuman CD44std FITC AHS4418 100 testsHuman CD44std Biotin AHS4419 100 testsHuman CD44v5 Unconj. AHS4451 100 µg/1 mLHuman CD44v5 Biotin AHS4459 100 testsHuman CD44v6 Unconj. AHS4461 100 µg/1 mLHuman CD44v6 Biotin AHS4469 100 testsHuman CD44v6 FITC AHS4468 100 testsHuman CD44v7 Unconj. AHS4471 100 µg/1 mLHuman CD44v7-v8 Unconj. AHS4481 100 µg/1 mLHuman CD44v10 Unconj. AHS4491 100 µg/1 mLHuman CD44 Unconj. AHS4441 200 µL

Product Description Part Number SizeHuman CD18 Unconj. AHS1801 1 mg/0.5 mLHuman CD18 FITC AHS1808 100 tests/1 mLHuman CD18 R-PE AHS1807 100 tests/1 mLHuman CD19 Unconj. AHS1911 200 tests/2 mLHuman CD19 Azide-free AHS1912 100 µg/100 µLHuman CD19 R-PE AHS1917 100 testsHuman CD19 FITC AHS1918 100 testsHuman CD19 R-PE (F(ab')2TEST™) AHM7013 100 tests/2x1 mLHuman CD19 Unconj. (F(ab')2TEST™) AHM7016 100 tests/2x1 mLHuman CD20 Unconj. AHS2001 200 tests/2 mLHuman CD20 Azide-free AHS2002 100 µg/100 µLHuman CD20 FITC AHS2008 100 tests/1 mLHuman CD21 Unconj. AHS2101 200 tests/2mLHuman CD21 Azide-free AHS2102 100 µg/100 µLHuman CD21 FITC AHS2108 100 tests/1 mLHuman CD21 R-PE AHS2107 100 tests/1 mLHuman CD22 Unconj. AHS2201 200 tests/2 mLHuman CD22 FITC AHS2208 100 tests/1 mLHuman CD22 R-PE AHS2207 100 tests/1 mLHuman CD23 Unconj. AHS2301 200 tests/2 mLHuman CD23 Azide-free AHS2302 100 µg/100 µLHuman CD23 FITC AHS2308 100 tests/1 mLHuman CD23 R-PE AHS2307 100 tests/1 mLHuman CD24 Azide-free AHS2402 200 µg/1 mLHuman CD25 Azide-free AHS2512 100 µg/100 µLHuman CD25 Biotin AHS2519 100 tests/1 mLHuman CD25 FITC AHS2518 100 tests/1 mLHuman CD25 R-PE AHS2517 100 tests/1 mLHuman CD26 Unconj. AHS2611 200 testsHuman CD27 Ascites AHS2701 200 tests/2 mLHuman CD28 Unconj. AHS2801 200 testsHuman CD28 FITC AHS2808 100 testsHuman CD28 Azide-free AHS2812 100 µg/100 µLHuman CD28 FITC AHS2818 100 tests/1 mLHuman CD29 Unconj. AHS2901 200 tests/2mLHuman CD29 Azide-free AHS2902 100 µg/100 µLHuman CD29 FITC AHS2908 100 tests/1 mLHuman CD30 Unconj. AHS3002 200 tests/2 mLHuman CD31 Unconj. AHS3102 200 µgHuman CD31 FITC AHS3108 100 tests/1 mLHuman CD31 R-PE AHS3107 100 tests/1 mLHuman CD32 Unconj. AHS3201 200 µgHuman CD33 Azide-free AHS3312 200 µgHuman CD33 FITC AHS3318 100 tests

Product Description Part Number SizeHuman CD56 Azide-free AHS5602 100 µg/100 µLHuman CD57 Unconj. AHS5702 100 µgHuman CD58 Unconj. AHS5801 200 µgHuman CD58 FITC AHS5808 100 tests/1 mLHuman CD59 Purified AHS5902 200 µgHuman CD59 FITC AHS5908 100 testsHuman CD61 Unconj. AHS6101 200 µg/200 µLHuman CD61 FITC AHS6108 100 tests/1 mLHuman CD61 R-PE AHS6107 100 tests/1 mLHuman CD62E Unconj. AHS6211 100 µg/1 mLHuman CD62E Biotin AHS6219 100 testsHuman CD62E FITC AHS6218 100 testsHuman CD62L Unconj. AHS6231 100 µg/1 mLHuman CD62L Biotin AHS6239 100 testsHuman CD62L FITC AHS6238 100 testsHuman CD64 Supernatant AHS6401 200 tests/2 mLHuman CD66b FITC AHS6628 100 tests/2 mLHuman CD68 Unconj. AHS6801 100 testsHuman CD71 Unconj. AHS7122 100 µg/100 µLHuman CD71 FITC AHS7128 100 tests/1 mLHuman CD72 Unconj. AHS7211 200 µgHuman CD74 Purified AHS7402 200 µgHuman CD76 Azide-free AHS7601 100 µg/100 µLHuman CD76 FITC AHS7608 100 tests/1 mLHuman CDw78 Unconj. AHS7801 200 tests/2 mLHuman CDw78 Unconj. AHS7811 100 µg/100 µLHuman CD79a FITC AHS7918 100 testsHuman CD87 uPA - Receptor AHS8702 100 µgHuman CD90 AHU0052 200 tests/2 mLHuman CD90 R-PE AHU0057 100 tests/1 mLHuman CD90 FITC AHU0058 100 tests/1 mLHuman CD95 AHS9552 100 µgHuman CD95 Unconj. AHS9502 100 µg/1 mLHuman CD95 Unconj. AHS9522 100 µg/1 mLHuman CD95 Azide-free AHS9542 100 µg/100 µLHuman CD95 FITC AHS9538 100 testsHuman CD99R Ascites AHS9911 0.2 mLHuman CD102 Unconj. AHT0201 50 µg/1 mL

Product Description Part Number SizeHuman CD44 (v7-v8) AHS4481 100 µgHuman CD45 Unconj. AHS4552 100 µg/100 µLHuman CD45 FITC AHS4558 100 tests/2 mLHuman CD45 R-PE AHS4557 100 tests/2 mLHuman CD45RA Unconj. AHS4521 200 tests/2 mLHuman CD45RA Azide-free AHS4522 100 µg/100 µLHuman CD45RA FITC AHS4528 100 tests/1 mLHuman CD45RA R-PE AHS4527 100 tests/1 mLHuman CD45RB Unconj. AHS4561 200 µgHuman CD45RC Unconj. AHS4531 200 tests/0.5 mLHuman CD45RO Unconj. AHS4541 200 tests/2 mLHuman CD45RO FITC AHS4548 100 tests/1 mLHuman CD45RO R-PE AHS4547 100 tests/1 mLHuman CD47 Unconj, AHS4702 200 µg/200 µLHuman CD48 Unconj. AHS4802 200 µgHuman CD48 R-PE AHS4807 200 µgHuman CD48 FITC AHS4808 100 tests/1 mLHuman CD49b Unconj. AHS4921 200 µg/200 µLHuman CDw49d FITC AHS4948 100 tests/1 mLHuman CDw49d Unconj. AHS4941 200 µgHuman CD49f Unconj. AHS4961 2 mLHuman CD49f FITC AHS4968 100 tests/1 mLHuman CD49f R-PE AHS4967 100 tests/1 mLHuman CD49c AHS4971 200 µg/2 mLHuman CD50 Azide-free AHS5002 100 µg/100 µLHuman CD50 FITC AHS5008 100 tests/1 mLHuman CD50 Azide-free AHS5012 100 µg/100 µLHuman CD51 Supernatant AHS5101 200 µg/2 mLHuman CD51 AHS5111 200 µg/2 mLHuman CDw52 Unconj. AHS5201 200 tests/0.5 mLHuman CD52 AHS5292 200 µgHuman CD52 FITC AHS5298 100 tests/1 mLHuman CD53 Unconj. AHS5301 200 tests/2 mLHuman CD54 Azide-free AHS5432 100 µgHuman CD54 Azide-free AHS5422 100 µg/100 µLHuman CD54 Biotin AHS5429 100 tests/1 mLHuman CD54 FITC AHS5428 100 testsHuman CD54 R-PE AHS5427 100 testsHuman CD54 Unconj. AHS5411 50 µg/1 mLHuman CD54 Biotin AHS5419 100 testsHuman CD54 FITC AHS5418 100 testsHuman CD54 (ICAM-1) AHS5444 0.5 mgHuman CD55 Unconj. AHS5501 200 µg/200 µLHuman CD55 Blocking Unconj. AHS5511 200 µgHuman CD56 Unconj. AHS5601 200 tests/2 mL

Monoclonal Antibodies to Rat Leukocyte CD AntigenProduct Description Part Number SizeRat CD2 purified ARS0203 1 mg/1 mLRat CD4 Unconj. ARS0401 1.0 mgRat CD4 Biotin ARS0409 100 testsRat CD4 FITC ARS0408 1 mg/1 mLRat CD4 R-PE ARS0407 100 testsRat CD5 Unconj. ARS0501 250 µLRat CD5 Biotin ARS0509 100 testsRat CD5 FITC ARS0508 1 mg/1 mLRat CD5 R-PE ARS0507 100 testsRat CD8 Unconj. ARS0801 1.0 mgRat CD8 Biotin ARS0809 100 testsRat CD8 FITC ARS0808 1.0 mgRat CD8 R-PE ARS0807 100 testsRat CD11b Unconj. ARS1121 1 mg/1 mLRat CD11b FITC ARS1128 1 mg/1 mLRat CD11b R-PE ARS1127 100 testsRat CD25 Unconj. ARS2502 0.5 mgRat CD25 Biotin ARS2509 100 testsRat CD25 FITC ARS2508 1.0 mgRat CD25 R-PE ARS2507 100 testsRat CD25 (NDS 61) Ascites ARS2513 1 mg/1 mLRat CD26 Supernatant ARS2601 2 mLRat CD43 Unconj. ARS4301 1 mg/1 mLRat CD43 Biotin ARS4309 100 testsRat CD43 FITC ARS4398 100 testsRat CD43 R-PE ARS4307 100 testsRat CD44 Supernatant ARS4401 200 µg/2 mLRat CD45 Unconj. ARS4501 0.5 mgRat CD45 Biotin ARS4509 100 testsRat CD45 FITC ARS4508 1.0 mgRat CD45 R-PE ARS4507 100 testsRat CD45RA Unconj. ARS4511 1 mg/1 mLRat CD45RA Biotin ARS4519 100 testsRat CD45RA FITC ARS4528 100 testsRat CD45RA R-PE ARS4517 100 testsRat CD45RC Ascites ARS4531 250 µLRat CD45RC FITC ARS4538 1 mg/1 mLRat CD53 Ascites ARS5301 250 µLRat CD71 Purified ARS7102 0.5 mg

Product Description Part Number SizeHuman CD102 Biotin AHT0209 100 testsHuman CD102 FITC AHT0208 100 testsHuman CD106 Supernatant AHT0611 2.0 mLHuman CD106 FITC AHT0608 100 testsHuman CD106 R-PE AHT0607 100 testsHuman CD106 Supernatant AHT0601 200 µgHuman CD115/c-fms/CSF1R AHT1502 100 µgHuman CD115/c-fms/CSF1R AHT1512 100 µgHuman CD117/c-kit/SCF Recombinant AHT1702 100 µgHuman CD117/c-kit/SCF Recombinant AHT1712 100 µgHuman CD117/c-kit/SCF Recombinant AHT1722 100 µgHuman CD120a AHR3011 100 µgHuman CD130 AHT3001 100 µgHuman CD132 AHT3201 100 µgHuman CD137 AHT3702 100 µgHuman CD138 Azide Free AHT3802 100 µgHuman CD138 R-PE AHT3807 100 testsHuman CD138 FITC AHT3808 100 testsHuman CD148 Unconj. AHT4802 100 µgHuman CD154 AHT5402 200 µgHuman CD154 FITC AHT5408 100 testsHuman CD155 AHT5502 100 µg

Monoclonal Antibodies to Miscellaneous Human Cell Surface MarkersProduct Description Part Number SizeHuman HLA Class II (DR) Azide-free AHU0182 0.1 mg/0.1 mLHuman HLA Class II (DR) FITC AHU0188 100 testsHuman HLA Class II (DR) R-PE AHU0187 100 testsHuman NKB1 Unconj. AHU0191 100 µgHuman Fas Ligand AHU0212 100 µgHuman Fas Ligand AHU0219 100 testsHuman NGFR AHU0302 100 µg

Monoclonal Antibodies to Mouse Leukocyte CD AntigenProduct Description Part Number SizeMouse CD3 Supernatant AMS0301 0.25 mgMouse CD3 FITC AMS0308 100 tests/1 mLMouse CD3 Unconj. AMS0311Z 0.1 mgMouse CD3 Unconj. AMS0311X 0.5 mgMouse CD3 Azide-free AMS0312Z 0.1 mgMouse CD3 Azide-free AMS0312X 0.5 mgMouse CD3 Biotin AMS0319 0.25 mgMouse CD3 FITC AMS0318 0.25 mgMouse CD4 Supernatant AMS0401 2 mLMouse CD4 FITC AMS0408 100 tests/1 mLMouse CD4 Biotin AMS0419 100 testsMouse CD5 Supernatant AMS0501 2 mLMouse CD5 R-PE AMS0507 100 testsMouse CD8α Azide-free AMS0812Z 0.1 mgMouse CD8a Azide-free AMS0812X 0.5 mgMouse CD8α FITC AMS0818 0.25 mgMouse CD8a R-PE AMS0817 0.1 mgMouse CD8α Supernatant AMS0801 0.25 mgMouse CD8a FITC AMS0808 100 tests/1 mLMouse CD8α R-PE AMS0807 100 tests/1 mLMouse CD8a Biotin AMS0809 100 tests/1 mLMouse CD11a Unconj. AMS1131 0.2 mgMouse CD11a Biotin AMS1139 100 testsMouse CD11a FITC AMS1138 100 testsMouse CD11a R-PE AMS1137 100 testsMouse CD11b Supernatant AMS1121 0.5 mgMouse CD11b FITC AMS1128 100 tests/1 mLMouse CD11b R-PE AMS1127 100 tests/1 mLMouse CD11b Unconj. AMS1191Z 0.1 mgMouse CD11b Unconj. AMS1191X 0.5 mgMouse CD11b Azide-free AMS1192Z 0.1 mgMouse CD11b Azide-free AMS1192X 0.5 mgMouse CD18 Supernatant AMS1801 200 tests/2 mLMouse CD19 Unconj. AMS1901 0.2 mgMouse CD19 Biotin AMS1909 100 testsMouse CD19 FITC AMS1908 100 testsMouse CD19 R-PE AMS1907 100 testsMouse CD44 Azide-free AMS4402 0.25 mg/0.25 mLMouse CD44 Unconj. AMS4411 200 µgMouse CD44 Biotin AMS4419 100 testsMouse CD44 FITC AMS4418 100 tests

Rat Miscellaneous Cell Surface AntibodiesProduct Description Part Number Size Rat T-Cell Receptor Unconj. ARU0011 0.25 mg/0.5 mLRat T-Cell Receptor Biotin ARU0019 100 testsRat T-Cell Receptor FITC ARU0018 1 mg/1 mLRat T-Cell Receptor R-PE ARU0017 100 testsRat Pan T-cells ARU0022 0.5 mgRat Thy1.1 Unconj. ARU0032 250 µLRat/Mouse Thy 1.1 Purified ARU0043 1 mg/1 mLRat/Mouse Thy 1.1 Biotin ARU0049 100 testsRat/Mouse Thy 1.1 FITC ARU0048 1 mg Rat/Mouse Thy 1.1 R-PE ARU0047 100 testsRat peripheral T-Cells Supernatant ARU0051 2 mLRat RT1Aa Ascites ARU0061 0.25 mLRat RT1Aa Purified ARU0073 1 mg/1 mLRat RT1Aa FITC ARU0078 1 mgRat RT1Ac Ascites ARU0081 0.25 mLRat RT1Au Supernatant ARU0271 5.0 mLRat RT1B Supernatant ARU0101 5 mLRat/Mouse RT1B Unconj. ARU0111 1 mg/1 mLRat/Mouse RT1B Biotin ARU0119 100 testsRat/Mouse RT1B FITC ARU0118 1 mg/1 mLRat/Mouse RT1B R-PE ARU0117 100 testsRat/Mouse RT1Bu Unconj. ARU0121 0.25 mLRat/Mouse RT1Bu Biotin ARU0129 100 testsRat/Mouse RT1Bu FITC ARU0128 1 mgRat/Mouse RT1Bu R-PE ARU0127 100 testsRat RT1Bac Supernatant ARU0131 2 mLRat RT1D Unconj. ARU0142 1 mg/0.2 mLRat Monocytes and Macrophages Ascites ARU0151 0.25 mLRat Monocytes and Macrophages FITC ARU0158 100 testsRat Thymocytes, Neurons, Endothelium,B-Cells and Follicular Dendritic CellsSupernatant ARU0161 2 mLRat Endothelium ARU0181 1.0 mgRat Activated Lymphocytes Supernatant ARU0201 2 mLRat Epidermal Langerhans Cells Ascites ARU0241 0.2 mgRat Dendritic Cells (Integrin) Supernatant ARU0251 2.0 mLRat Integrin Molecule Ascites ARU0261 0.2 mL

Product Description Part Number Size Mouse CD154 Unconj. AMT5411X 0.5 mgMouse CD154 Unconj. AMT5411Z 0.1 mgMouse CD154 Azide-free AMT5412X 0.5 mgMouse CD154 Azide-free AMT5412Z 0.1 mgMouse CD154 FITC AMT5418 0.25 mgMouse CD154 R-PE AMT5417 0.1 mg

Mouse Miscellaneous Cell Surface AntibodiesProduct Description Part Number Size Rat/Mouse Thy1.2 Ascites AMU0051 0.25 mLMouse Macrophages and Monocytes AMU0071 2 mLMouse Lymphocytes Subset Ascites AMU0091 0.5 mLMouse B-Cells Ascites AMU0111 0.5 mLMouse Vβ4 Unconj. AMU0121 2 mLMouse Vβ4 Biotin AMU0129 100 tests/0.5 mLMouse Vβ4 FITC AMU0128 100 tests/1 mLMouse Vα8 Unconj. AMU0131 2 mLMouse Vα8 Biotin AMU0138 100 testsMouse Vβ10 Supernatant AMU0141 2 mLMouse Vβ10 Biotin AMU0149 100 testsMouse Vβ11 Supernatant AMU0151 2 mLMouse Vβ11 Biotin AMU0159 100 testsMouse Vβ11 FITC AMU0158 100 testsMouse Vβ12 Supernatant AMU0271 2 mLMouse Thymic Epithelial CellsSupernatant AMU0171 2 mLMouse MHC Class II Supernatant AMU0191 5 mLMouse Ly-6A.2 Supernatant AMU0231 2 mLMouse Ly-6B.2 Ascites AMU0241 0.2 mLMouse Heat Stable Antigen Supernatant AMU0261 2 mLMouse B-Cell Antigen AMU0281 0.25 mg

Product Description Part Number Size Mouse CD44 R-PE AMS4417 100 testsMouse CD45 Unconj. AMS4501 200 µgMouse CD45 Biotin AMS4509 100 testsMouse CD45 FITC AMS4508 100 testsMouse CD45 R-PE AMS4507 100 testsMouse CD45R Unconj. AMS4511Z 0.1 mgMouse CD45R Unconj. AMS4511X 0.5 mgMouse CD45R Azide-free AMS4512Z 0.1 mgMouse CD45R Azide-free AMS4512X 0.5 mgMouse CD45R FITC AMS4518 0.25 mgMouse CD48 Supernatant AMS4801 2.0 mLMouse CD49d Unconj. AMS4941 200 µgMouse CD49d Biotin AMS4949 100 testsMouse CD49d FITC AMS4948 100 testsMouse CD49d R-PE AMS4947 100 testsMouse CD54 Unconj. AMS5403 0.1 mgMouse CD62L Unconj. AMS6211Z 0.1 mgMouse CD62L Unconj. AMS6211X 0.5 mgMouse CD62L Azide-free AMS6212Z 0.1 mgMouse CD62L Azide-free AMS6212X 0.5 mgMouse CD62L Biotin AMS6219 0.25 mgMouse CD62L FITC AMS6218 0.25 mgMouse CD62L R-PE AMS6217 0.1 mgMouse CD71 Supernatant AMS7102 0.25 mgMouse CD80 Unconj. AMS8011Z 0.1 mgMouse CD80 Unconj. AMS8011X 0.5 mgMouse CD80 Azide-free AMS8012Z 0.1 mgMouse CD80 Azide-free AMS8012X 0.5 mgMouse CD80 R-PE AMS8017 0.1 mgMouse CD80 Unconj. AMS8001 50 µgMouse CD86 Unconj. AMS8601X 0.5 mgMouse CD86 Unconj. AMS8601z 0.1 mgMouse CD86 Azide-free AMS8602X 0.5 mgMouse CD86 Azide-free AMS8602Z 0.1 mgMouse CD86 FITC AMS8608 0.25 mgMouse CD86 R-PE AMS8607 0.1 mgMouse CD106 Unconj. AMT0601 200 µgMouse CD106 Biotin AMT0609 100 testsMouse CD106 FITC AMT0608 100 testsMouse CD106 R-PE AMT0607 100 tests

Monoclonal Antibodies to Rabbit Cell Surface Antigens Product Description Part Number SizeRabbit CD4 ALS0401 2 mLRabbit CD5 ALS0502 0.2 mgRabbit CD9 ALS0901 2 mLRabbit CD11a ALS1101 2 mLRabbit CD11b ALS1121 2 mLRabbit CD18 ALS1801 2 mLRabbit CD43 ALS4301 2 mLRabbit CD44 ALS4401 2 mLRabbit CD45 ALS4502 0.2 mgRabbit CD58 ALS5801 2 mLRabbit Thymocytes, T-Cells, Neutrophils,Platelets ALU0011 2 mLRabbit MHC Class I ALU0031 2 mLRabbit MHC Class II ALU0041 2 mLRabbit IgM (B-Cells) ALU0051 2 mL

Monoclonal Antibodies to Monkey Leukocyte CD AntigenProduct Description Part Number SizeMonkey CD3 Unconj. APS0301 0.5 mgMonkey CD3 Biotin APS0309 200 TestsMonkey CD3 FITC APS0308 100 TestsMonkey CD3 R-PE APS0307 100 Tests

Monoclonal Antibodies to Guinea Pig Cell Surface AntigensProduct Part Number SizeGuinea Pig CD4 Ascites AGS0401 0.25 mLGuinea Pig CD8 Ascites AGS0801 0.25 mLGuinea Pig T-Lymphocytes Ascites AGU0011 0.25 mLGuinea Pig T-Lymphocytes TCS AGU0021 2 mLGuinea Pig T-Cell Subset TCS AGU0031 2 mLGuinea Pig B-Cell Subset TCS AGU0041 2 mLGuinea Pig Pan B-Cells AGU0051 2 mLGuinea Pig Lymphocytes (HomingReceptor), Langerhans Cells Ascites AGU0061 0.25 mLGuinea Pig Lymphocytes, LangerhansCells TCS AGU0071 2 mLGuinea Pig MHC Class II Supernatant AGU0092 1.0 mLGuinea Pig Macrophages and MonocytesAscites AGU0101 0.25 mLGuinea Pig Fibronectin TCS AGU0111 2 mLMouse Anti Human (cross-reactive with Guinea Pig)Keratin Type I Purified AGU0131 2 mLKeratin Type II Purified AGU0141 2 mL

Monoclonal Antibodies to Bovine Cell Surface AntigensProduct Description Part Number SizeBovine CD1 Ascites ABS0101 0.25 mLBovine CD2 Ascites ABS0201 0.25 mLBovine CD4 Ascites ABS0401 0.25 mLBovine CD5 Ascites ABS0501 0.25 mLBovine CD6 Ascites ABS0601 0.25 mLBovine CD8 Ascites ABS0801 0.25 mLBovine CD14 Ascites see m x sheep CD 14Bovine CD41 TCS ABS4101 2 mLBovine CD43 Ascites see m x sheep CD 43Bovine CD45 Ascites ABS4501 0.25 mLWorkshop Cluster 1 ABU0011 0.25 mL

Monoclonal Antibodies to Sheep Cell Surface Antigens Product Description Part Number SizeSheep CD1c TCS AOS0101 2 mLSheep CD8 TCS AOS0801 2 mLSheep CD14 TCS AOS1401 2 mLSheep CD18 TCS AOS1801 2 mLSheep CD31 TCS AOS3101 2 mLSheep CD43 TCS AOS4301 2 mLSheep CD45 TCS AOS4501 2 mLSheep Macrophage (42/44 kDa) TCS AOU0011 2 mLSheep Macrophage (75-80 kDa) TCS AOU0021 2 mLSheep MHC Class I TCS AOU0031 2 mLSheep MHC Class II-bTCS AOU0041 2 mLSheep Lambda TCS AOU0051 2 mL

Intracellular Staining

IntroductionCytokines are secreted regulatory proteins that control the maintenance, growth, differ-entiation and effector function of the immune system and tissue cells. Released cytokinesinteract with their target cells which are often adjacent to the producer cells, resulting inthe appropriate biological response. Frequently, producer cells secrete very small quanti-ties of cytokine. The role of cytokines in immunological processes, hematopoiesis, inflam-matory responses and the interaction between different biological pathways can be bet-ter understood by studying the processes involved in cytokine production within their cellof origin.

One of the major applications of Intracellular Staining is to study cytokine production inspecific peripheral blood mononuclear cells (PBMCs) in response to various mitogens.The study of intracellular cytokine production has often been hampered by methods thatrely on the measurement of released cytokines within a mixed population of cells, suchas sandwich ELISAs. Other methods which measure cytokine messenger RNA, such as insitu hybridization, Northern blotting and RNase Protection Assays, are time consumingand labor intensive and often do not reflect the amount of protein expressed within thecell. Intracellular Staining, however, provides a rapid and simple method of measuringcytokine production in specific cell populations using flow cytometry.

PurposeIn this technique, a cell population is stimulated to produce cytokine. Stimulations arecarried out in the presence of the transport inhibitor, monensin, which prevents therelease of cytokines into the extracellular milieu. The stimulated cells are stained with flu-orescein isothiocyanate (FITC) tagged anti-CD monoclonal antibody and fixed withparaformaldehyde. Following this, cells are stained with a R-Phycoerythrin (R-PE) labeledanti-cytokine antibody in the presence of a permeabilizing agent. The stained cells arethen analyzed by flow cytometry for the co-existence of the two different fluorescent tagsin the same cell.

With the technique of Intracellular Staining, there is one major caveat in data analysis. Ifno signal is observed within a specific cell population, researchers typically presume thatthe cytokine of interest is not produced in these cells. However, the same result isobtained when the anti-cytokine antibody is not gaining access into the internal environ-ment of the cell. With this in mind, our kit provides researchers with a novel permeabi-lization control, Perm-a-Sure™. This control is a FITC-labeled antibody which yields a dis-tinct pattern when cells are unpermeabilized, partially permeabilized and fully perme-abilized. This easy, one-step control, which can be incorporated into every experimentalrun, ensures accurate data analysis for both novice and experienced users.

B.

D

CD62L R-PE

B

CD3 FITC

A

CD28 R-PE

CD45R FITC

C

Figure 3: Mouse splenocytes from different animals were labeled with:

3a: Anti-Mouse CD3 FITC (AMS0318)

3b: Anti-Mouse CD28 R-PE (AMS2807)

3c: Anti-Mouse CD45R FITC (AMS4518)

3d: Anti-Mouse CD62L R-PE (AMS6217)

Figure 3

+45%

Monoclonal Antibodies to Canine Cell Surface AntigensProduct Part Number SizeCanine CD4 Pure ADS0401 1 mgCanine CD5 Pure ADS0501 1 mgCanine CD8 Pure ADS0802 0.25 mgCanine CD11/18 TCS ADS1101 2 mLCanine CD44 TCS ADS4402 2 mLCanine CD45 Pure ADS4501 1 mgCanine CD45R Ascites ADS4532 2 mLCanine Thy 1 Purified ADS0012 0.25 mgCanine Thy 1 TCS ADS0021 2 mLCanine MHC Class II Pure ADS0032 0.25 mgCanine Claw Cluster H TCS ADS0041 2 mL

Table 3:

Intracellular Staining:1. Aliquot the fixed cells into tubes at a density of 106/tube or process together and

wash 2 times in 1 mL ICPerm™.2. Spin the cells at 300 x g for 5 minutes and aspirate the supernatant.3. Resuspend cells in 40 µL of ICPerm™.4. Add anti-human cytokine antibody*R-PE (typically 0.1-0.5 µg/106 cells) and incu-

bate at 4ºC for 30 minutes. Increasing the amount of antibody during this incuba-tion can increase non-specific background so it is best to start at the lower rangeand optimize from there.

5. Spin cells at 300 x g for 5 minutes and wash 2 times in 1 mL ICPerm™.6. Wash cells one more time in PBS alone and resuspend in 0.5 mL 50 mM phosphate

buffered saline, pH 7.3, for flow cytometric analysis. At least one of the followingspecificity controls is recommended: a) appropriate isotype control (for example, Mouse IgG1*R-PE, Catalog #AML2318);b) pre-incubating conjugated antibody with recombinant human cytokine (refer to

catalog); c) pre-blocking cells with unconjugated anti-human cytokine (see Figure 4d, page 33)

prior to staining with conjugated antibody.

Special Note: Add 2% BSA or other non-specific protein such as normal mouse serumto reduce non-specific signal.

Procedure for Staining PBMCs with an Anti-Human Cytokine R-PE Conjugate:

MaterialsCytokine monoclonal antibody Isotype control (FITC conjugate) (optional)

(R-PE conjugate) Phosphate buffered saline (PBS), pH 7.3Isotype control (R-PE conjugate) PBS/0.1% sodium azide/1%FCSPerm-a-Sure™ (FITC conjugate) Vortex mixerICFix™ fixation buffer Benchtop centrifugeICPerm™ permeabilization buffer AspiratorICBlock™ cell transport inhibitor Flow Cytometer (calibrated)Cell surface Monoclonal antibody Fluorescent microscope (optional)

(FITC) (optional) 2% BSA or normal serum

A complete listing of reagents begins on page 34.

Note: All buffers are shown in bold type, formulations are shown in Table 2, below.

Isolation and Stimulation of Human PBMCs:PBMCs are isolated from whole blood by Ficoll-Paque density gradient separation. Thecells are stimulated to increase the cytokine of interest. The stimulant and time for stim-ulation are dependent on the cytokine of interest and should be investigated prior to cellset-up. Typically cells can be stimulated with PMA/Ionophore for lymphocytic cytokinesor chemokines such as Interferon-γ, IL-4, IL-2, etc. For monocytic cytokines orchemokines such as IL-6, IL-8, TNF-α, IL-10, etc., cells can be stimulated with 1 µg/mLLPS. The time for stimulation can vary from 4-48 hours. Stimulation should be carriedout in the presence of a protein transport inhibitor to prevent the release of the proteinfrom the cell. Monensin (Catalog #PNN0011) (2 µM final) or Brefeldin A (1µg/mL) canbe used. Either monensin or Brefeldin A or a combination of both work well and shouldbe optimized for your particular cytokine. See Table 3 (next page) for specific conditions.

Cell Fixation:1. After stimulation of the cells, wash 2 times with PBS/sodium azide/FCS and stain

with appropriate cell surface staining markers (e.g., CD3*FITC, CD4*FITC,CD8a*FITC, CD14*FITC, see page 15).

2. After washing an additional 2 times with PBS/sodium azide/FCS, resuspend the cellsin ICFix™ and incubate at 4ºC for 10 minutes.

3. Wash an additional 2 times with PBS/sodium azide/FCS. The fixed cells can bestored at 4°C for up to 7 days prior to intracellular staining.

Stimulations for Intracellular CytokinesCytokine Stimulant(s) Dose(per mL media) Time Temp. (°C) %CO ICBlock

GM-CSF PMA+Calciumionophore A23187 50 ng+250 ng 44 hours 37 5 +

Interleukin-1α LPS 1 µg 6 hours 37 5 +Interleukin-1β LPS 1 µg 6 hours 37 5 +Interleukin-2 PMA+Calcium

ionophore A23187 50 ng+250 ng 5-6 hours 37 5 +Interleukin-3 PMA+Calcium

ionophore A23187 50 ng+250 ng 6-20 hours 37 5 +Interleukin-4 PHA or 10 µg or 4-6 hours 37 5 +

PMA/ionophore 50 ng+250 ngInterleukin-5 PMA+Calcium

ionophore A23187 50 ng+250 ng 5-6 hours 37 5 +Interleukin-6 LPS 1 µg 5-6 hours 37 5 +Interleukin-8 PMA+Calcium

ionophore A23187 50 ng+250 ng 5-6 hours 37 5 +Interleukin-10 LPS 1 µg 18 hours 37 5 +Interleukin-12 LPS+PMA 1 µg+50 ng 5-6 hours 37 5 +Interleukin-13 PMA+Calcium

ionophore A23187 50 ng+250 ng 6-8 hours 37 5 +Interferon-γ PMA+Calcium

ionophore A23187 50 ng+250 ng 5-6 hours 37 5 +TNF-α PMA+Calcium

ionophore A23187 50 ng+250 ng 5-6 hours 37 5 +or LPS or 1 µg

Table 2: Buffers:ICFix™ (Cat. #FB001): 4% paraformaldehyde

50 mM phosphate buffered saline, pH 7.3(Catalog #309-500)

ICPerm™ (Cat. #PB001): 50 mM phosphate buffered saline, pH 7.31% (v/v) fetal calf serum (Catalog #200C-100)0.1% (w/v) sodium azide and0.1% (w/v) saponin

These buffers are also available as a convenient, ready to use kit (Catalog #ANN0001).

Antibodies for Flow Cytometry-Intracellular StainingTypical Data for Intracellular Staining of Rat IL-6 and IL-2.

Human Antibodies for Intracellular StainingProduct Description Part Number SizeAnti h GM-CSF AHC2012 0.5 mgAnti h GM-CSF R-PE AHC2017 0.1 mgAnti h IFN-γ AHC4332 0.5 mgAnti h IFN-γ FITC AHC4338 0.1 mgAnti h IFN-γ R-PE AHC4337 0.1 mgAnti h IL-1α AHC0312 0.5 mgAnti h IL-1β FITC AHC0918 0.1 mgAnti h IL-1β R-PE AHC0917 0.1 mgAnti h IL-2 AHC0422 0.5 mgAnti h IL-2 FITC AHC0428 0.1 mgAnti h IL-2 R-PE AHC0427 0.1 mgAnti h IL-3 AHC0732 0.5 mgAnti h IL-3 R-PE AHC0737 0.1 mgAnti h IL-4 AHC0642 0.5 mgAnti h IL-4 FITC AHC0648 0.1 mgAnti h IL-4 R-PE AHC0647 0.1 mgAnti h IL-5 AHC0954 0.5 mgAnti h IL-5 FITC AHC0958 0.1 mgAnti h IL-5 R-PE AHC0957 0.1 mgAnti h IL-5 AHC0852 0.5 mgAnti h IL-6 AHC0762 0.5 mgAnti h IL-6 FITC AHC0768 0.1 mgAnti h IL-6 R-PE AHC0767 0.1 mgAnti h IL-8 AHC0682 0.5 mgAnti h IL-8 FITC AHC0688 0.1 mgAnti h IL-8 R-PE AHC0687 0.1 mgAnti h IL-10 AHC0102 0.5 mgAnti h IL-10 FITC AHC0108 0.1 mgAnti h IL-10 R-PE AHC0107 0.1 mgAnti h IL-12 AHC9122 0.5 mgAnti h IL-12 R-PE AHC0127 0.1 mgAnti h IL-13 AHC0132 0.5 mgAnti h IL-13 R-PE AHC0137 0.1 mgAnti h TNF-α AHC3912 0.5 mgAnti h TNF-α FITC AHC3918 0.1 mgAnti h TNF-α R-PE AHC3917 0.1 mg

Figures 4a & 4b: Rat macrophages were stimulated with 1 µg/mL LPS in the presence of 1 µL ICBlockTM

and 1 µg/mL Brefeldin A. After 12 hours, the cells were harvested, washed and fixed with ICFixTM.

After washing, the cells were permeabilized using ICPermTM buffer with 2% BSA. The cells were

aliquoted at 106 cells/tube and incubated with 10 µL of anti-rat macrophage FITC (Catalog

#ARU0158) and 100 ng of mouse anti-rat IL-6-R-PE (Catalog #ARC0967) (figure 4a). The upper-right

hand quadrant shows that 65% of the macrophages produce IL-6 when stimulated by LPS. To

demonstrate that this increase is specifically due to the stimulant, unstimulated cells were stained with

these antibodies using the same procedure (figure 4b).

Figures 4c & 4d: Rat splenocytes were stimulated with PMA and calcium ionophore (50 ng/mL and

250 ng/mL, respectively) in the presence of 1 µL ICBlock™ and 1 µg/mL Brefeldin A. After 6 hours,

the cells were harvested, washed and fixed with ICFixTM. The cells were then permeabilized using

ICPermTM buffer with 2% BSA. The cells were aliquoted at 106 cells/tube and incubated with 100 ng

of mouse anti-rat IL-2-R-PE (Catalog #ARC0927) (figure 4c). The upper-left hand quadrant shows that

9% of the rat splenocytes produce IL-2 when stimulated with PMA and ionophore. To demonstrate the

specificity of staining, the stimulated rat splenocytes were incubated with 10 µg unconjugated anti-

rat IL-2 (Catalog #ARC0922) prior to the addition of the R-PE conjugated antibody (figure 4d).

anti-

rat

IL-2

R-P

E

autofluoresence FITC

Figure 4c Figure 4d

anti-

rat

IL-2

R-P

E

autofluoresence FITC

Figure 4b

anti-

rat

IL-6

R-P

E

anti-rat macrophage FITC

Figure 4a

anti-rat macrophage FITCan

ti-ra

t IL

-6 R

-PE

Immunohistochemistry

Introduction

Immunohistochemistry is used to identify the location and distribution of target antigensin cells or tissues by staining with a specific antibody. The antibody is conjugated to eithera fluorescent or colorimetric label, and the location of the label seen through a micro-scope approximates the position of the target antigen. We present here methods for stain-ing of cervical tissue, brain tissue, and various cell lines.

Procedure I:

Purpose: Procedure for indirect staining of cytokines in PFA fixed cryostat sectionswith monoclonal antibodies

Extracted from Walid Al-Saleh Thesis (ULG, Pr. Boniver Service of Anatomo-Pathology), Liege,Belgium. Detection of IL-2 , IL-4 , IL-6 , IL-10 , IL-12 , IFN-γ and TNF-α proteins in cervical biop-sies were made by an original immunohistology technique.

Materials and EquipmentFrozen sections of sample tissues Refrigerator (4°C)Glass slides Primary antibodyCoverslips Secondary antibody4% Paraformaldehyde, pH 7.4 Diaminobenzidine tetrachlorideTris-buffered Saline, 0.1% Saponin, Avidin/Biotin/Peroxidase

pH 7.4 (Vectastain, Vector Labs)Tris-buffered Saline/0.3% H2O2/0.1% Hematoxylin

Saponin, 0.02% NaN3 Ethanol1% Goat Serum in TBS/Saponin Microscope

Procedure:1. Dry frozen tissue sections of 8 µm thickness at room temperature for 2 hours.2. Fix sections with 4% paraformaldehyde for 15 minutes at room temperature.3. Wash slides 2X, 5 minutes each, with TBS/0.1% saponin.4. Block endogenous peroxidase by incubating 30 minutes in TBS/H2O2/saponin/NaN3.5. Wash slides 3X, 3 minutes each with TBS/saponin.6. Block non-specific binding sites with 1/100 diluted goat serum in TBS/saponin for

20 minutes.7. Incubate overnight at 4°C, with the appropriate antibody.8. Wash slides 4 times in TBS/saponin, incubate the slides with biotinylated secondary

antibody for 30 minutes.9. Add avidin-biotin-peroxidase, and reveal the resulting peroxidase activity by incu-

bating the slides with a 0.5 mg/mL solution of DAB, prepared in distilled water,which produces a brown precipitate at the level of the antigen-primary antibody.

Rat Antibodies for Intracellular StainingProduct Description Part Number SizeAnti r IL-2 R-PE ARC0927 0.1 mgAnti r IL-2 FITC ARC0928 0.1 mgAnti r IL-4 R-PE ARC0947 0.1 mgAnti r IL-10 R-PE . ARC9107 0.1 mgAnti r IL-10 FITC ARC9108 0.1 mg

Mouse Antibodies for Intracellular StainingProduct Description Part Number SizeAnti m/rat IFN-γ ARC4033 1.0 mgAnti m/rat IFN-γ FITC ARC4038 0.1 mgAnti m/rat IFN-γ R-PE ARC4037 0.1 mgAnti m IL-2 AMC0022 0.5 mgAnti m IL-2 FITC AMC0028 0.1 mgAnti m IL-2 R-PE AMC0027 0.1 mgAnti m IL3 Unconj. AMC0034 0.5 mgAnti m IL-3 FITC AMC0038 0.1 mgAnti m IL-3 R-PE AMC0037 0.1 mgAnti m IL-4 AMC0042 0.5 mgAnti m IL-4 FITC AMC0048 0.1 mgAnti m IL-4 R-PE AMC0047 0.1 mgAnti m IL-6 AMC0862 0.5 mgAnti m IL-6 R-PE AMC0867 0.1 mgAnti m IL-12 AMC0124 0.5 mgAnti m IL-12 FITC AMC0128 0.1 mgAnti m IL-12 R-PE AMC0127 0.1 mgAnti m TNF-α AMC3012 0.5 mgAnti m TNF-α FITC AMC3918 0.1 mgAnti m TNF-α R-PE AMC3917 0.1 mg

Intracellular Staining ReagentsProduct Description Part Number SizeICFix™/ICPerm™ Buffer Kit ANN0001 100 testsICFix™ Buffer FB001 100 testsICPerm™ Buffer PB001 100 testsICBlock™ PNN0011 100 testsPerm-a-Sure™

Permeabilization Control AHU0208 20 tests

Protocol A: Used with prostate epithelial cells

1. Plate cells on glass cover slips and allow to attach overnight.2. Rinse 1X with PBS.3. Fix the cells with formaldehyde/PBS for 15 minutes.4. Wash 1X with PBS.5. Treat with Triton X-100 for 2 minutes.6. Wash with 1X PBS.7. Incubate with primary antibody (diluted in PBS) for at least 1 hour.8. Wash 3X for 5 minutes per wash using PBS.9. Incubate with the secondary antibody, goat anti-rabbit Ig’s*FITC (Catalog #ALI4408),

(diluted in PBS) for 1 hour.10. Wash 3X for 5 minutes per wash using PBS.11. Mount each cover slip on a slide using GelMount (Fisher).12. Analyze by fluorescence microscopy.

Protocol B: Used with NIH3T3 and F111 Cells

1. Starve cells by incubating in serum-free medium for 24 hours.2. Stimulate cells with an appropriate stimulus (e.g., 10% serum, EGF or PDGF in

DMEM for 0-60 minutes (e.g., can do a time course of 0, 5,10,15, 30, and 60 minutes).3. Fix cells with 10% paraformaldehyde in PBS for 15 minutes.4. Rinse fixed cells 3X with PBS.5. Do a peroxidase block step by incubating with 1 mL of 30% peroxide in 100 mL

of PBS for 30 minutes.6. Permeabilize the cells with 100% methanol by incubating for 10 minutes at -20°C.7. Rinse cells 3X with PBS.8. Block with 1% BSA for about 30-60 minutes.9. Rinse 3X with PBS.10. Incubate with primary polyclonal antibody (300 ng/mL-1 µg/mL) for 1 hour in PBS

containing 5% goat serum and 0.25% NP40. 11. Wash 3X with PBS.12. Incubate with secondary antibody (e.g., goat anti-rabbit Ig’s, Catalog #ALI4405) at

1:2000 in 2% goat serum in PBS.13. Wash 3X with PBS.14. Conjugate Vector ABC (Vectastain Elite) kit for 30 minutes and then dilute to

20 µL/mL for each reagent in PBS (as indicated by the supplier).15. Add ABC reagents for 60 minutes.16. Wash 3X with PBS.17. Add DAB substrate (one tablet of each per 1 mL).

Protocol C: Used with Rat52, Swiss 3T3, NIH3T3 and 293 cells

1. Fix cells in 10% paraformaldehyde (15 minutes at RT).2. Permeabilize in 100% methanol (10 minutes at -20°C).3. Block with 1% BSA.4. Incubate with the primary polyclonal antibody (at 300 ng/mL) for 1 hour.5. Wash and add fluorescein-labeled secondary, goat anti-rabbit Ig’s* FITC (Catalog

#ALI4408).

10. Wash slides in TBS 4 times.11. Counterstain for 1 minute with hematoxylin.12. Dehydrate slides with sequential ethanol washes of 1 minute each starting with

75%, followed by 80% and finishing with a 100% ethanol wash.13. Seal slides.14. Analyze by optical microscopy.

Note: It is not necessary to add detergent to the TBS buffer. The protocol can be fol-lowed as is with saponin omitted from all buffers when staining CDs.

A complete listing of cytokine antibodies can be found on page 34.

Procedure II:

Purpose: Procedures for indirect staining of intracellular phosphate residues.

As illustrated in the section on Western blotting (Chapter 4), the use of phosphorylationstate-specific antibodies is highly useful for demonstrating whether a particular residue ina protein is phosphorylated. Another critical question is to determine the subcellularlocalization of the phosphorylated protein. In many cases, phosphorylation of proteinscan result in redirecting a protein to a new location in the cell (e.g., membrane, focaladhesions, nucleus etc.), thereby determining the nature and consequence of the signaltransduction pathways that occur. The following protocols have been used successfully byacademic scientists to validate phosphorylation state-specific polyclonal antibodies.Importantly, the extensive purification procedure used to generate these polyclonal anti-bodies, including the resulting high avidity, provides high signal-to-noise ratios in thisapplication.

A complete listing of phosphorylation state specific antibodies can be found on page 60.

Materials and EquipmentSample cells/Stimulant 30% H2O2

Glass slides and coverslips 1% BSAPBS Primary Antibody 3.7% & 10% formaldehyde in PBS Secondary Antibody10% paraformaldehyde GelMount0.2% Triton X-100 Fluorescent MicroscopeMethanol Goat serum0.06% TritonX/PBS

Immunoprecipitation/Western Blotting

Cells grown in culture or in the context of a physiological tissue respond to a variety ofextracellular stimuli including growth factors, hormones, cytokines, chemokines, extra-cellular matrix factors/integrin ligation, bioactive peptides and a variety of stress stimuli(e.g., UV light, osmotic or heat shock, viral infection, inhibitors of transcription or trans-lation etc.). In many cases, the activation of individual cells involves interaction of a stim-ulus with a cell surface receptor, which in turn, transmits a signal to the cell interior.Indeed, signal transduction is the process whereby a cell generates a chemical changethat alters the activity or location of one or more proteins inside the cell. It is the sum ofthese changes that determines the ultimate physiological response to a series of physio-logical stimuli. Thus the same principal applies whether one is stimulating adipocyteswith insulin, which binds to and activates the insulin receptor, to study aspects of diabetesor studying the effect of beta amyloid peptides on isolated neurons as a part of unravel-ing the defects that lead to Alzheimer’s Disease.

A large variety of proteins are activated as part of signal transduction pathways. These caninclude cell surface receptors, protein kinases, protein phosphatases, phospholipases,GTP hydrolases, cytoskeletal/microfilament proteins, transcription factors, and otherproteins involved in homeostasis, metabolism, DNA replication, RNA transcription andRNA translation. Protein phosphorylation on one or more serine, threonine, or tyrosineresidues within a protein, is a very common post-translational modification that regulatesmost cellular processes. This modification requires that a particular protein kinase rec-ognize a target sequence (containing Ser, Thr, or Tyr residues), within a protein.Conversely, a protein phosphatase is an enzyme capable of removing the phosphate moi-ety from a particular Ser, Thr, or Tyr residue within this sequence, thereby setting up adynamic balance between protein phosphorylation and dephosphorylation. Many of theantibodies made by BioSource International are made in a manner so as to specificallydetect the presence of a phosphorylated residue within a target sequence of a protein ofinterest. These phosphorylation state-specific antibodies allow sensitive and selectivequantitation of a phospho-epitope and thereby provide valuable insight as to the regula-tion of a particular signal transduction pathway.

The detailed protocols that follow illustrate different approaches to each immunologicalmethod that have been successfully used by in-house R&D scientists or which were pro-vided by external scientists who have worked extensively with our antibodies. These pro-tocols are applicable to monoclonal and polyclonal as well as phospho-specific or ‘pan’antibody products. Proper handling of the antibodies and a willingness to systematicallyvary conditions for a given protocol are two very important factors that can greatly affectthe results obtained with any antibody preparation.

Antibodies that recognize total target protein (referred to as ‘pan’ antibodies for lack of abetter term), will detect both the phosphorylated and non-phosphorylated forms of thetarget protein. These antibodies are useful to detect the total amount of target protein, ofwhich a fraction may be phosphorylated. The second group of antibodies is the phos-

Note: Peak activation may vary with the cell type and/or the target enzyme. For example,MAPK/ERK1 & 2 activation following mitogen stimulation generally occurs around 5 min-utes, where the enzyme is predominantly located in the cytoplasm. After 10-15 minutessome fraction of the enzyme is translocated to the nucleus. By 20 minutes, the MAPKenzymes are generally turned off presumably due to the action of various phosphatases.

Protocol D: Used with Rat Brain Slices

This is a protocol used on 30 µm floating rat brain sections. The rats had been perfusedwith Ringer's followed by 4% paraformaldehyde. Tissues were dissected and post-fixedovernight in the same fix. The next day, tissues were rinsed in PBS and cryoprotectedovernight in 30% sucrose. The following day, frozen sections were cut and placed in PBSin 48 well plates (add: 0.02% sodium azide if they are not going to be used immediate-ly). For immunostaining: all steps are at room temperature with shaking unless indicated.

1. Wash 3 X 5 minutes in PBS.2. Permeabilize in 0.06% Triton X-100/PBS for 30 minutes.3. Wash 3 X 5 minutes in PBS.4. Block with 3% normal serum in PBS (goat serum if secondary is a goat antibody)

for 30 minutes.5. Incubate in primary antibody diluted in 3% normal serum in PBS overnight at 4°C

(Try broad dilution series initially with test antibody and control antibody).6. Next day wash 3 X 5 minutes in PBS.7. Incubate in secondary antibody diluted in PBS (usually 1:500 or 1:1000 will be

suitable for most secondary antibodies) for 1 hour.8. Wash 3 X 5 minutes in PBS.9. For fluorescent secondary antibodies, mount sections in Vectashield (+ DAPI if a

nuclear marker is desired); for HRP-conjugated secondary antibodies or (biotin-streptavidin-HRP procedures) add DAB substrate, develop until color is visible(~10 min), and mount in 100% glycerol or Fluormount G.

Table 4:

TroubleshootingProblem: Solution:High background -add 0.1% Tween 20 to washes

-increase normal serum (5-10%) for blockingNo specific staining -try different fixations, e.g., Bouin’s, glutaraldehyde, or

10% formalin-microwave sections to increase antigen exposure-vary antibody concentration, incubation times, and

wash times

Stimulation of Cells

1. Activating Mitogen Activated Protein Kinase Pathway

2. Activating Stress-activated Pathway

Cell culture conditions prior to activation:

1. PC12 (rat pheochromocytoma) cells are grown in RPMI 1640 medium with 10% horseserum, 5% Fetal Bovine Serum, 25 mM HEPES, 0.5 mM EGTA, and favorite antibiotic(s)on a bed of collagen (coat containers with rat tail collagen type I at 6 µg/cm2).

2. Stimulate cells with anisomycin, sorbital or NGF-withdrawal to activate the stress-

phorylation state-specific antibodies. These antibodies are used to detect the level of tar-get protein that has been phosphorylated at the designated amino acid residue (i.e., eitherphosphorylated on serine, threonine, or tyrosine residues). In contrast to generic anti-phosphotyrosine, phosphoserine, or phosphothreonine antibodies, which recognizephosphorylation at all sites in a protein, use of antibodies to individual phosphorylationsites in the context of the surrounding peptide sequence provides important informationabout differential phosphorylation/dephosphorylation of different sites within a protein.Specificity for the phosphorylated protein of interest derives from careful choice of thepeptide sequence surrounding the phosphorylated residue, based on multiple sequencealignments using one or more sequence databases. In most cases, specificity has beendemonstrated using peptide competition experiments (see Appendix) or by analysis ofsite-directed mutants where the phosphorylated residue of interest has been changed to anon-phosphorylatable residue (e.g., Tyr -> Phe or Ser/Thr -> Ala).

In this section you will find procedures outlining the stimulation of cells, extraction ofprotein, immunoprecipitation and immunoblotting of phosphorylated proteins. The stim-ulations include activations of either stress pathways or mitogen activated protein kinasepathways. Extractions are described either with the use of detergent, requiring an ultra-centrifuge, or without detergent. An assay for protein content is also briefly described.Immunoprecipitation protocols address isolation of either phosphorylated proteins or cellcycle proteins. The protocols given for immunoblotting have been validated using eitherpan antibodies or phosphorylation-state specific antibodies. Before proceeding, pleasereview the protocols carefully to select the ones which are most suited to your investiga-tions.

Purpose: Preparation of Cells for Detection of Phosphorylation State of Proteins.

General Guidelines

1. Grow the cells of interest as recommended by the ATCC datasheet or other sourcefor that particular cell line.

2. Count the cells by trypan blue exclusion and plate the cells equally for the numberof time-points or stimulations required in 100 mm dishes. Larger dishes can be usedif more cells are required.

3. Prior to treating the cells, the serum should be reduced to 5% for 24 hours thenreduced further to serum-free media for at least 10-12 hours before adding stimu-lant. This will prevent any interference from serum factors on the phosphorylationstate of the proteins in the signaling cascade. If the cells are dependent on any otherfactors such as cytokines they must also be removed for 10-12 hours prior to stim-ulation. An alternate method for serum starvation is to reduce the serum to 0.5%from 5% for 18-24 hours then reducing to serum-free medium for 2 hours prior tostimulation.

4. Place in 37ºC incubator until ready to use.

293, CHO, 3T3, and U373 cells:For cell culture conditions: 293 (human embryonic kidney), CHO (Chinese ham-

ster ovary), 3T3 (mouse fibroblasts), U373 (human astrocytes), rat 1 HIRc B (rat fibro-blasts that overexpress the human insulin receptor) cells are grown in Dulbecco'sModified Eagles Medium (DMEM, Catalog #104-500) containing 10% fetal bovineserum (Catalog #200P-100) and 1% L-glutamine.

For stimulation of cells to activate MAPK pathway: Prior to stimulation these cellsare washed once with serum-free medium and incubated in the same medium for 16-18 hours. Cells are then incubated with fresh serum-free medium for 5 minutes at 37°Ceither without activator (basal control plates) or with the addition of EGF (50 ng/mL),TPA (100 nM), insulin (100 nM) or FBS (10%).

All cell lines are then washed once with cold phosphate buffered saline andextracts are prepared as follows.

PC12 cells:For cell culture conditions: PC12 (rat pheochromocytoma) are grown in RPMI

1640 medium with 10% horse serum (Catalog #201-100), 5% fetal bovine serum, 0.2 mMEGTA (molar equivalent to Ca2++) and 15 mM HEPES on a bed of collagen (coat 150mm dishes with rat tail collagen type I at 6 µg/cm2).

For stimulation of cells to activate MAPK pathway: Cells should be near conflu-ence but not packed. PC12 cells should be flat and not rounded up (this is facilitatedby use of EGTA or using a more behaved clone like PC12w cells). These cells areexposed to fresh FBS-containing medium with EGTA for ~18 hours prior to treatmentwith NGF (50 ng/mL) for 5 minutes at 37°C.

All cell lines are then washed once with cold phosphate buffered saline andextracts are prepared as follows.

induced enzymes (p38 and JNK).3. Add fresh medium the day before harvesting.

Preparation of Cell Extracts

1. Without detergent (requires ultracentrifugation):

-Used successfully with MAPK/ERK, SAPK and p38 signaling pathway.

Materials and EquipmentD-PBS (Catalog #309-500) Homogenization BufferIce CentrifugeHomogenizer UltracentrifugeFreezer (-80°C) Refrigerator (4°C)Liquid N2

Table 5:

1. Remove media.2 Wash cells with D-PBS.3. Scrape the cells into iced Homogenization Buffer.4. Break the cells on ice using 30-50 strokes of a homogenizer (Note: perform all sub-

sequent steps at 4°C).5. Centrifuge the homogenates at 4000 x g for 5 minutes and collect the supernatants.6. Centrifuge the supernatants at 97,000 x g for 60 minutes.7. Freeze the final supernatants in liquid nitrogen and store aliquots at -80°C.

2. With detergent:

This has been shown to work successfully with our phosphorylation-state specific anti-bodies. In this protocol, an ultracentrifugation step is not necessary.

-Used successfully with Pyk2 and FAK signalling pathway.

Materials and EquipmentPBS without Ca++ and Mg, 4°C IceCentrifuge Vortex mixerFreezer (-80°C) Extract Buffer50 mL conical tubes

Table 6:

1. Stimulate cells in desired way with time points if necessary. Cells should be stim-ulated in serum-free media.

2. Add ice cold PBS without calcium or magnesium (PBS) to stop stimulation. Placecells immediately on ice and remove buffer. If suspension cells, add ice cold PBS,spin at 4ºC for 5 minutes at 300 x g, then remove buffer.

3. Wash cells this way in ice cold PBS two more times.4. For adherent cells, add 10 mL of ice cold PBS and scrape cells on the ice. Put

into 50 mL conical tubes and bring volume to 50 mL, pellet cells and resuspendin appropriate volume of Extract Buffer. Typically 500-1000 µL of ExtractBuffer is used to resuspend pellet or 5 x 106 cells.

5. Place cells in microfuge tubes on ice. Vortex extract vigorously every 2 minutesfor 10 minutes, leaving the extract on ice in between times.

6. Centrifuge the extract at 14,000 x g for 15 minutes at 4ºC.7. Remove supernatant and discard pellet. Freeze supernatant at -80ºC until ready to

use. It is better to aliquot cells since freeze thawing can destroy or interfere withthe phosphorylation state of the protein.

Reagent: Extract Buffer: 10 mM Tris, pH 7.4100 mM NaCl1 mM EDTA1 mM EGTA1 mM NaF20 mM Na4P2O72 mM Na3VO41% Triton X-10010% glycerol0.1% SDS0.5% deoxycholate1 mM PMSF (stock is 0.3 M in DMSO)Protease inhibitor cocktail from Sigma (madeaccording to manufacturer’s guidelines)(add 250 µL per 5 mL extract buffer)

This buffer is good for 2-3 weeks at 4ºC or aliquoted (without protease inhibitors andPMSF added) at -20ºC for 6 months (thaw on ice). Important: Add the protease inhibitors just before using. Stability = 24 hours at 4ºC. PMSF is very unstable and must be added prior to use, even if added previously.

Reagents for Stimulation 2.Anisomycin (Sigma): use a 10 mg/mL stock solution to make the medium

10 µg/mL anisomycin for 30 minutes before harvest.

Sorbitol (Sigma): use a 2.5 M stock solution to make the medium 0.5 Msorbitol 5 minutes before harvest.

NGF-withdrawal: grow the cells an additional 9 to 10 days in DMEMplus 0.5 mM EGTA, 1% horse serum, 50 ng/mLNGF (Catalog #PHG0124) and favorite antibiotic(s). Then,wash cells once with medium without NGF. Addmedium without NGF but with an anti 2.5S NGFantibody (Promega) at 100 µg per two 75 cm2 flasks.Incubate an additional 5 to 6 hours, then harvest.

Reagents:Homogenization Buffer20 mM Tris-HCl, pH 7.520 mM p-nitrophenyl phosphate (general phosphatase inhibitor)1 mM EGTA (Ca++ chelator)50 mM sodium fluoride (Ser/Thr phosphatase inhibitor)50 µM sodium orthovanadate (Tyr phosphatase inhibitor)5 mM benzamidine (protease inhibitor)

Immunoprecipitation

Purpose: To separate the target antigen from a complex mixture of cellular componentsusing a target specific antibody for the purpose of purification, quantification, or identifi-cation.

Use of a pan antibody for immunoprecipitation prior to Western blotting is recommend-ed if a protein is expressed at low levels or if the amount of phosphorylated protein is sus-pected to be low.

1. Immunoprecipitation Protocol I: Validated using phosphorylation-state specific anti-bodies to retain the phosphorylation state and kinase activity.

Materials and EquipmentLysis Buffer 2X Sample BufferMicrocentrifuge Incubator/Water Bath (95°C)Primary antibody 32PProtein A Sepharose SubstrateRocker P81 FiltersRefrigerator (4°C) Scintillation counter

Table 7:1. Stimulate cells using appropriate stimulation method (see pages 42-43) and solubi-

lize cells in Lysis Buffer (20 minutes at 4°C).2. Spin in microfuge at 20,000 x g for 10 minutes.3. Determine protein concentration of extracts (page 45).4. Adjust protein concentration to 0.5 mg/mL.5. Incubate 0.5 mg of extract with 1 µg of the primary polyclonal antibody for 2 hours

at 4°C with continuous rocking.6. Add 25 µL of protein A sepharose suspension (Pharmacia); incubate 1 hour at 4°C

with continuous rocking.7. Spin in microfuge (5 seconds), and wash pellet 3X with Lysis Buffer.

Protein Assay for Cell Extracts

Before an IP or Western can be run on the extract, the protein concentration of the frac-tions needs to be assessed by a Bradford Assay. This can be done by using the Bio-Radprotocol and using frozen BSA standards.

Materials and EquipmentBio-Rad Protein Assay Kit Microtiter plates1 mg/mL BSA Spectrophotometer

BioRad Protein Assay Standard Procedure for Microtiter Plates:1. Prepare protein standards; 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg/mL BSA in water.2. Prepare dye reagent by diluting 1 part Dye Reagent Concentrate with 4 parts

deionized, distilled water. 3. Prepare the unknown protein solutions.4. Protein concentrations will vary; however, dilutions of 1:2 to 1:10 in water typi-

cally result in concentrations that will fall within the specificity range of the assay.5. Pipettte 10 µL of each standard and diluted unknown sample solution into sepa-

rate microtiter plate wells.6. Add 200 µL of diluted dye reagent to each well. Mix the samples and reagent

thoroughly, using a microplate mixer. At this point, you should be able to visuallydetermine whether the unknown dilutions used are within the specifcity range ofthe assay. If visually within the range, proceed to Step 7; if not, dilute theunknowns accordingly and add 10 µL to wells, then proceed to Step 6.

7. Incubate at room temperature for at least 5 minutes. Absorbance will increaseover time; samples should incubate at room temperature for no more than 1 hour.

8. Measure absorbance at 595 nm. Configure the template, accounting for dilutions,and label the assay, read the plate, and analyze the data curvalinearly.

Continue Westerns or Immunoprecipitations on the samples according to the followingprotocols.

Reagents:2X Sample Buffer: 125 mM Tris, pH 6.8

(SB) 4% SDS, 20% Glycerol10% β-Mercaptoethanol0.0025% Bromphenol Blue

Lysis Buffer: 20 mM Tris-HCl, pH 7.41% NP40 (non-ionic detergent)137 mM NaCl50 µM EDTAprotease inhibitors (PMSF, aprotinin,Leupeptin) as needed

8. Rotate for ≥ 30 minutes at 4ºC.9. Spin for 5 seconds at RT and discard the supernatant. 10. Wash beads 4 times with 500 µL cold Extract Buffer.11. Vortex, spin for 5 seconds at RT and discard the supernatant. 12. Resuspend beads in 10-30 µL of 2X SB and mix well. (For non-denatured samples,

prepare Sample Buffer without SDS and freeze beads after addition of buffer.)13. Heat to ≥ 95º for 4-5 minutes.14. Spin for 1-2 minutes at RT to pellet the beads.15. Run supernatant on gel. (Supernatant can be frozen at ≤ -20ºC for future use.)16. Follow Western blot procedure if desired; for IP/Western (see page 50).

For SDS-PAGE:a) Wash pellet 1X with water.b) Resuspend in 2X Sample Buffer (SB)c) Heat at 95°C for 2 minutes.d) Spin in microfuge, remove supernatant and store in labeled tube.e) Repeat addition of SB and combine supernatants.f) Run PAGE

For Kinase Assays:a) Wash pellet in 1X reaction buffer.b) Resuspend in reaction buffer and 2 µCi of 32P-ATP and Myelin basic protein

as a substrate.c) Spot reaction onto P81 filters, wash and quantitate by scintillation counting.

Figure 5

2. Immunoprecipitation Protocol II: Validated using cell cycle antibodies to retain pro-tein complexes and kinase activity.

1. Prepare protein extracts in Extract Buffer (see page 44).2. Prepare Protein A/G beads by washing beads in Extract Buffer 3 times to equili-

brate.3. Determine protein concentration of extracts (page 45).4. Use 200-500 µg of total protein from cell extracts.5. Add approximately 1.0 µg of antibody. A typical antibody to extract ratio is 1-5 µg

to 500 µg extract. (You can “preclear” the extract by incubating for 30 minutes at4ºC with an aliquot of Protein A/G beads, if desired, prior to this step).Alternatively, Protein G can be incubated with antibody for 30 minutes at 4ºCprior to addition of precleaned cell lysate.

6. Rotate tube for at least 1 hour at 4ºC.7. Add 50-60 µL of Protein A/G beads. Beads should be at 1:1 volume with buffer.

IP/Western of rat testes cell extractsusing anti-STAT4 (44-366).

STAT4 ➛

IgG ➛

Western Blot

Several procedures outlining Western blotting are given below. Specific details for per-forming both colorimetric and chemiluminescent detection are provided. In most cases,cell or tissue extract samples (1-30 µg of total protein) can be analyzed by conventionalSDS-PAGE using mini-gels with a 10-12 well comb format.

Procedure I: This has been validated using our pan antibodies with either recombinantor native proteins.

1.1 Antigen PreparationIn a microcentrifuge tube, dilute antigen to 20 ng/µL with PBS, if using recombinant orpurified protein. Alternatively, if cell extracts are being used, pipette amount of cellsneeded into clean centrifuge tube, typically 1-30 µg cell lysate is required per lane.Further dilute sample with an equal volume of either 2X nonreducing electrophoresissample buffer or 2X reducing electrophoresis sample buffer. If 2X reducing sample bufferis used, boil samples for 5 minutes.

Materials and Equipment (we recommend the Novex system)10%, 14%, or 16% , 4-20% Novex Tris-Glycine precast gels2 sponges (approximately 11 cm2)25 cm2 container, or largerElectrophoresis equipment (mini-gel)*MicrocentrifugePower supplyPrestained molecular weight marker (Novex) Primary antibodyPolyvinylidene Fluoride (PVDF) membrane (Fisher)RockerWeigh boatsSecondary antibody, IgG, Alkaline Phosphatase conjugate (See section table for

appropriate antibody selection)Western blotting apparatusWhatman #1 filter paperStaining and Destaining Solutions Developing solution (BioRad Alkaline Phosphatase Conjugate Substrate Kit for colori-

metric developing or Tropix Western CDP-Star Ready-to-Use Substrate Solution)

* It is recommended that investigators follow the manufacturer’s instructions for the apparatus used.

Note: All buffers are shown in bold type, formulations are on page 53.

1.2 Polyacrylamide Gel Electrophoresis (PAGE)1. Set up NOVEX gel in electrophoresis chamber with 1X Running Buffer for PAGE.2. Use 10% gel for proteins > 30 kDa; 14% gel for proteins ≤ 30 kDa; and 16% gels

for proteins ≤10 to 4 kDa. Alternatively, a gradient gel of 4-20% may be used forcell extracts.

Figure 6: Immunoprecipitation illustration

Table 8:

1.4 Staining Procedure (optional)

1. Remove the PVDF membrane from apparatus and place in large weigh boat. If trans-fer was successful, the prestained molecular weight markers should now be on PVDFmembrane, not on the gel. The higher molecular weight standards (blue and orangebands) are more difficult to transfer, so some of the color may still be present on thegel. This is all right if the rest of the bands have transferred onto the PVDF.

2. Rinse the PVDF membrane with 10 mL tTBS for 5 minutes, 3 times. Membranescan be dried at this point for storage.

3. To store membranes: Once transfer is complete, wash membrane with wash bufferand put membrane into Amido Black stain solution for 30 seconds.

4. To destain, place in Destain Solution until protein bands are clearly visible andmembrane is not too dark.

Table 9:

3. Load 10 µL prestained molecular weight markers.4. Load sample : For recombinant protein, load 20 µL of antigen into appropriate

lanes/wells. For cell extracts, typically load 1-30 µg of cell extract/lane or 200-500 µgof cell extract on a one lane gel.

5. Run gel at 120 volts for 10 minutes, followed by 130 volts for 90 minutes, until thedye front is at the bottom of the resolving gel.

6. Remove gel from cast.7. With a razor blade, remove dye front from bottom of gel and remove loading lanes

at top of gel.

1.3 Immunoblotting1. Before PAGE is finished running, cut PVDF membrane to size slightly larger than that

of the gel. Bookmark one edge where the top of lane one will be represented.

Note: Wear gloves whenever handling PVDF membrane.

2. Soak membrane in methanol for 1 minute, then rinse with diH2O for 5 minutes.3. Cut 2 pieces of Whatman filter paper slightly larger than size of the gel (At least as

large as the membrane).4. Gather together 2 sponges, the 2 pieces of Whatman paper, and the PVDF mem-

brane and soak in Transfer Buffer for 2 minutes prior to setting up apparatus.5. Lay electroblotting apparatus in a container that is at least 25 cm2 containing 250 mL

Transfer Buffer.6. Open the sandwich apparatus with the black side down in the container.7. Make a sandwich. Start by laying a sponge on the one side, then layering in the fol-

lowing order:

8. It is important that no bubbles form between the PVDF membrane and the gel.9. Close the sandwich apparatus making sure that the clasps are shut tight.10. Pour 1 liter of Transfer Buffer into tank and place sandwich apparatus in one of the

slots of the tank. (Use the cassette position nearest the center.)11. Connect the black electrode to the terminal nearest the black side of the sandwich

apparatus. The red electrode should be placed in the other terminal. This ensuresthat the proteins run from the gel onto the membrane towards the red terminal.

12. Run the gel at 140 mA with the voltage at the highest point (500V) for 60-90 min-utes at room temperature. This may require longer time if more than 2 gels are beingtransferred. Put transfer apparatus into container with ice.

13. Prepare the Wash Buffer (tTBS)14. Prepare Western Blocking Buffer.

Buffers:Transfer Buffer: 2.4 g Tris base

14.2 g Glycine200 mL MethanolQ.S. to 1 Liter and add 1 mL 10% SDS, cooledto 4°C prior to use

5X Running Buffer: 15 g Tris base72 g Glycine5 g SDSAdjust pH to 8.8 and Q.S. to 1 Liter. Dilute 1:4for 1X Running Buffer

Wash Buffer (tTBS): 9.68 g Tris base32 g NaClQ.S. to 4 liters. Adjust to pH 7.6 and add 2 mLTween-20

Western Blocking Buffer: 5 g fat-free dried milk100 mL Wash Buffer

Amido Black stain solution:1 g Amido Black100 mL methanol100 mL glacial acetic acidQ.S. to 1 L with diH2O

SpongeWhatman filter papergelPVDF membrane (smooth side facing gel)Whatman filter paperSponge

-

+

11. Pour 20 mL of Western Blocking Buffer onto PVDF membrane. Add 4 µL of sec-ondary antibody to the solution, for a 1:5000 dilution. (The amount of the second-ary antibody may be modified according to the specific experiment setup. TheTAGOIMMUNOLOGICALS™ secondaries have been shown to produce consistentresults between 1:5000-1:20,000 dilution.)

12. Place on rocker for 45 minutes to 1 hour at room temperature. Pour off secondaryantibody solution and rinse PVDF membrane with 10 mL Wash Buffer for 5 min-utes, with constant rocking.

13. Repeat wash step 3-4 more times.

1.5 Blot DevelopmentDuring final washing, prepare developing solution:

For Colorimetric Development:Combine the following:9.8 mL alkaline phosphatase developing buffer (0.1M Tris, pH 9.5)100 µL alkaline phosphatase Color Reagent A100 µL alkaline phosphatase Color Reagent BPour off Wash Buffer from final wash and add 10 mL developing solution.Incubate in developing solution until bands appear on membrane (usually between

2 and 10 minutes). If no bands appear in this time, incubate for 30 minutes.Pour off developing solution and rinse with 20 mL of diH2O 2 times for 5 minutes.Air dry and laminate.

For ECL Development:a) Carefully remove membrane from washing solution, sliding it along the side of

the dish to remove excess Wash Buffer (DO NOT let membrane dry!).b) Place membrane in a small dish or large weigh boat. Add 3-5 mL of CDP-Star

Ready-to-Use substrate solution (Tropix) from kit and incubate for 5 minutes.c) Remove membrane from dish by sliding it along side to remove excess solution.

Place it on a sheet of plastic film and carefully wrap. Gently rub out air bubbles.d) Expose blot to film for ≥15 seconds. Exposure times may vary according to the

antibody.e) Develop film.f) Dispose of blot unless otherwise required for further testing. If retained, store

blot in plastic film at 4°C. (Note: the signal will be stable for up to 18 hours after incubation with substrate.)

We have found that the preceeding protocol gives low background and clean signal whenused with our primary antibodies. Sometimes the protocol will have to be modifieddepending on the primary antibody used, the amount of target protein present in theextract, whether the phosphorylation state of the protein is being determined and the wayin which the extract was prepared. Procedure II has been used successfully with anti-bodies that showed high background or where no signal was detected with Procedure I.In addition, for guidelines helpful in optimizing low signal for Western blotting, see theAppendix, page 67.

Table 10:

5. At this point, membrane can be dried and stored for up to 1 year at room tempera-ture. If using right away, don’t dry. Wash membrane several times in Wash Buffer.If using dried membrane, wet in 100% methanol before using and wash with WashBuffer several times.

6. Place the PVDF membrane in 10 mL Western Blocking Buffer for at least 30 min-utes at room temperature, rocking constantly. This may be left overnight at 4°C withconstant rocking.

7. Pour out Western Blocking Buffer. Pour 10 mL of fresh Western Blocking Bufferonto PVDF membrane. Add 10 µL of primary antibody at 1 mg/mL to the solutionfor a 1:1000 dilution. Place on rocker for 1 to 2 hours at room temperature. (Theamount of the primary antibody may be modified according to the specific experi-ment setup, 1:500, 1:1000, or 1:2000.)

8. Pour off primary antibody solution and rinse PVDF membrane with 10 mL WashBuffer for 10 minutes, with constant rocking.

9. Repeat wash step 3-4 more times.10. Select the secondary antibody.

The secondary antibody is dependent upon the animal in which the primary anti-body is produced. For example, if the primary antibody is a mouse anti-human anti-body, the secondary antibody must be a goat anti-mouse IgG. Alkaline phosphataseconjugates are used for the colorimetric development and for the enhanced chemi-luminescence (ECL) development. (ECL is more sensitive than colorimetric andshould be used whenever possible.) See Table 11 below.

Table 11:

Destain Solution:100 mL glacial acetic acid100 mL isopropanolQ.S. to 1 L with diH2O

Primary Secondary (AP)Goat anti- Swine Whole Molecule Anti-Goat Ig’s, AP, Human Ig

Adsorbed (Catalog #ACI3405)Human anti- Goat F(ab’)2 Anti-Human IgG, AP, (Catalog #AHI1305)Mouse anti- Goat F(ab’)2 Anti-Mouse Ig’s, AP, Human Ig Adsorbed

(Catalog #AMI4705)Rat anti Goat F(ab’)2 Anti-Rat Ig’s, AP, Mouse and Human Ig

Adsorbed (Catalog #ARI4405)Rabbit anti- Goat F(ab’)2 Anti-Rabbit Ig’s, AP, Mouse and Human Ig

Adsorbed (Catalog #ALI4405)

2.1 Polyacrylamide Gel Electrophoresis (PAGE)

Note: Wear gloves when handling gels and nitrocellulose membranes.

Commercial 10% PAGE gels are rinsed with Nanopure water to eliminate azide storagebuffer.

1. Assemble SDS PAGE discontinuous gel apparatus and fill upper and lower bufferchambers.

2. In a microcentrifuge tube, mix protein samples with 2X Sample Buffer containingreducing agent. Note: Sample buffer containing DTT should be made fresh on theday of the experiment.

3. Heat samples for 2 minutes in 95°C heating block. 4. Spin samples for 5 seconds in a microcentrifuge and carefully load gel with each sample.5. Run the gel at 125V (constant voltage) until the dye front reaches the end of the gel

(approximately 1.5- 2 hours).

2.2 Immunoblotting

1. Remove gel from the apparatus and soak in transfer buffer for 15 minutes. While this isoccurring, cut nitrocellulose to fit the entire gel and 3 sheets of blotting paper. Soaknitrocellulose, blotting paper, and transfer apparatus sponges in transfer buffer as well.

2. Assemble transfer apparatus as follows, being careful not to introduce any air bubblesbetween layers:

Table 13:

3. Insert assembled blot into the blotting apparatus, with orientation of nitrocelluloseclosest to positive electrode (transferred proteins have net negative charge fromSDS). Add the ice finger pack and fill the tank with cold Western Transfer Buffer.

4. Transfer the entire assembled unit to 4°C and electroblot at 100 V for 1 hour.

2.3 Staining Procedure

1. Remove nitrocellulose blot and check that the prestained markers did indeed transfer.2. Block membrane with TBS + 1% BSA for a minimum of 1 hour at 37° or overnight

at 4°C.

Alternate Protocol with Phosphorylation State-Specific Antibodies

2. Procedure II: This has been validated using our phospho-state specific antibodies.

The supplied protocol is based on the use of Nitrocellulose membranes, an AlkalinePhosphatase (AP) labeled Goat Anti-Rabbit secondary antibody (Catalog #ALI4405), pre-qualified for use with the anti-polyclonal anitbody, and Western Blue Stabilized Substratefor alkaline phosphatase. Other procedures including the use of Nylon membranes (e.g.,PVDF, Immobilon, etc.) or other detection methods such as 125I-Protein A or non-radioac-tive/chemiluminescent detection using the AP secondary conjugate or HorseradishPeroxidase (HRP)-conjugated secondary antibodies can also be used. The cell extractsused below were prepared from pheochromocytoma (PC12) cells; however, similarresults have been obtained with many experimental systems including several cell lines(e.g., REF52, NIH3T3, Swiss3T3, F111, CHO, INS1 and 293 cells) and tissues (mam-malian muscle or brain and Drosophila).

Equipment:heating block at 95°C.electrophoresis unit (Hoeffer Mighty Small or equivalent)electroblotting unit (BioRad or equivalent)microcentrifuge (Eppendorf or equivalent)oscillating platform shaker (Labline or equivalent)power supply (capable of delivering 125V, constant voltage)

Table 12:

black half of apparatussponge1 sheet blotting papergelnitrocellulose paper1 sheet blotting paperspongewhite half of apparatus

-

+

Reagents:1x TBS Buffer:

Add 200 mL of 10x TBS to 1.8 L of nanopure water.TBST Buffer (TBS + 0.05% Tween-20):

Add 0.5 mL of Tween-20 to 1 liter of 1x TBS Buffer.Blocking Buffer (TBS +1% BSA):

Dissolve 1g of IgG-free and protease-free BSA (Jackson Laboratories) per 100 mL TBS.Antibody sample buffer (TBST +0.1% BSA):

TBST containing 0.1% BSA.1x Running Buffer:

24 mM Tris base0.19 M glycine10% SDS

1x Western Transfer Buffer :Mix 1000 mL Gel Transfer Buffer200 mL methanol700 mL deionized waterStore at 4°C!! Can be used twice before it needs to be replaced.

Gel Transfer Buffer:12 mM Tris base96 mM glycine10% SDS

7. Place the blot, protein side up and surrounded by plastic wrap, into the film cassettemaking sure that no detection solution leaks out into the film cassette since thefilm must remain dry.

8. Switch off the white light and work under the film-safe red lights. Carefully place asheet of autoradiography film (Kodak BioMAX Chemiluminescence film) on top ofthe blot, close the cassette and apply sufficient pressure to hold in place. Exposefor 15 seconds and do not move the film while it is being exposed.

9. Remove the film and develop in the automatic film developer.10. Place a second piece of film onto the blot, close the cassette and start a timer.11. On the basis of the appearance of the first piece of film following development,

estimate how long to continue the exposure of the second piece of film. Secondexposures typically vary from 30 to 60 seconds, depending on the intensity of thesignal.

12. Compare each experimental blot with the corresponding 'secondary conjugatealone control', especially if there are additional bands other than those correspon-ding to isoforms for the specific enzyme being detected.

Note: If background signals on the film are high, the blot can be rewashed twice for 10minutes with wash buffer and the detection steps 1-10 repeated.

3. Lay blot onto blotting paper and using a fresh razor blade, carefully cut the mem-brane into two strips.

4. Using Antibody Sample Buffer, dilute the primary antibody to 0.2-1 µg/mL in afinal volume of 20 mL. Transfer to a suitable container and add blot. In addition,probing replicate blots with a corresponding antibody that recognizes both activeand inactive forms (i.e., total enzyme) is a valuable control.

5. Using an oscillating platform shaker, incubate Blot #1 for 2 hours at ambient tem-perature.

6. Transfer and wash with wash buffer 3X 5 minutes (approximately 75 mL each wash).7. Dilute the Goat Anti-Rabbit Ig’s * AP (Catalog #ALI4405) conjugate 1:10,000 in

Antibody Sample Buffer and add to blot.8. Incubate 1 hour at ambient temperature using oscillating platform.9. Wash both blots with wash buffer 3X 5 minutes (approximately 75 mL each wash).10. Wash blots with TBS alone 2X 1 minute (approximately 30-40 mL each).11. Add both blots to plastic vessel containing Western Blue Stabilized NBT/BCIP.

Watch as bands begin to materialize.12. Stop color development by transferring strips to deionized water and rinse.

Note: NBT bands are stable as long as they are kept out of the light. Data can be pho-tographed via AMBIS for long term storage.

2.4 Chemiluminescent Detection Using the ECL Western blot Kit:

Notes: Read through this entire next section before proceeding since it is necessary towork quickly once the blots have been exposed to the detection solution. It is thereforehelpful to actually conduct all steps in the dark room so as to minimize any delays. Usegloves for this stage onward to prevent hand contact on film or detection reagents.Note that the color development will continue even after removing the blots from thedevelopment solution. Do not leave in solution too long as background will becomeincreasingly darker with time.

1. Prepare the ECL detection solution by mixing equal volumes of Solution 1 withSolution 2, as supplied with the ECL detection kit, to yield sufficient volume tocover the membrane (this requires ~ 0.125 mL/cm2 of membrane). Most standardminiblots require 0.5 mL of each solution (1 mL final volume). Mix the detectionsolution immediately after combining stock Solutions 1 and 2.

2. Add the detection solution directly to the blot on the surface to which the proteinwas transblotted. Do not allow the blots to dry out.

3. Incubate the membrane for precisely 1 minute at room temperature.4. Drain off excess detection solution by holding the blot vertical and touching the

edge of the blot against a piece of tissue paper.5. Gently place the blot, protein side down, on to a piece of plastic wrap. Fold over

the surrounding plastic wrap to form an envelope that encloses the blot. Avoidapplying excess pressure as this can cause high backgrounds.

6. Gently smooth out the air pockets. Again, avoid applying excess pressure as thiscan cause high backgrounds.

Phosphospecific Antibodies

Product Description Part Number SizeAkt/PKB [pT308] 44-622 10 BlotAkt/PKB [pS473] 44-602 10 BlotBad [pS112] 44-522 10 BlotBad [pS136] 44-524 10 BlotCannabinoid CB1 [pS316] 44-312 10 BlotCdk1 [pTpY14/15] 44-686 10 Blotc-Raf [pS621] 44-504 10 Blotc-Raf [pYpY340/341] 44-506 10 BlotCortactin [pY421] (mouse) 44-854 InquireEGFR [pY845] 44-784 10 BlotEGFR [pY1068] 44-788 10 BlotEGFR [pY1086] 44-790 10 BlotEGFR [pY1148] 44-792 10 BlotEGFR [pY1173] 44-794 10 BlotEGFR Sampler 44-799 7 mini-vialseIF-2α [pS51] 44-728 10 BloteIF-4E [pS209] 44-528 10 BlotERK1/2 [pTpY185/187] 44-680 10 BlotERK1/2 [pTpY185/187] FITC conj. 44-6808 10 BlotERK5/BMK1 [pYpY218/220] 44-612 10 BlotFAK [pY397] 44-624 10 BlotFAK [pY407] 44-650 10 BlotFAK [pY576] 44-652 10 BlotFAK [pY577] 44-614 10 BlotFAK [pS722] 44-588 10 BlotFAK [pY861] 44-626 10 BlotFAK [pS840] 44-590 InquireFAK [pS843] 44-592 InquireFAK [pS910] 44-596 10 BlotFAK [pY925] 44-616 10 BlotFAK [pY950] 44-598 InquireFAK Sampler 44-630 6 mini-vialsGSK-3β [pS9] 44-600 10 BlotGSK-3β[pY216]/α[pY279] 44-604 10 BlotIκBα [pSpS32/36] 44-726 10 BlotIntegrin beta-3 [pY773] 44-876 InquireIR/IGF1R [pY1158] 44-802 10 BlotIR/IGF1R [pYpY1162/1163] 44-804 10 BlotIRS-1 [pS616] 44-550 10 BlotJAK1 [pYpY1022/1023] 44-422 10 BlotJAK2 [pYpY1007/1008] 44-426 10 BlotJAK/STAT Sampler 44-430 7 mini-vialsJNK (SAPK) [pTpY183/185] 44-682 10 BlotLck [pS179] 44-842 InquireLck [pY505] 44-850 InquireMAPK Sampler 44-683 4 mini-vialsp38 [pTpY180/182] 44-684 10 Blotp53 [pS392] 44-640 10 Blot

Typical Data using Western blot Methods on Phophorylation-State Specific Antibodies

Figure 7 Figure 8

Figure 9 Figure 10

Pyk2

– +SDF-1α

Western blotting data using anti-Pyk2[pY402] (44-618) show the increasein Pyk2 phosphorylation in humanpro-B cells treated with100 µg/mL SDF-1α for 5 minutes.

116kD➝

CEF cell extracts were blottedwith 0.1 µg/mL anti-FAK [pY397](44-624).

125kD➝

Western blotting data usinganti-Src (44-656) at 1 µg/mL inhuman platelets.

60kD➝

Rat PC12 cell extracts were blottedwith 0.1µg/mL anti-ERK 1/2(44-654).

44kD➝42kD➝

NeutralizationIntroductionNeutralization allows determination of the potency or capacity of the antibody to inter-fere with the expected or normal biological activity of the target protein.

Purpose: For the determination of neutralization capability of rabbit anti-rat TNF-α: astested in the standard cytotoxicity assay used for assessing TNF-α biological activity.

Materials and EquipmentCells Microtiter plateAntibody, endotoxin-free CentrifugeCytokine, endotoxin-free Incubator (37°C)

Day 11. The target cells, adherent L929 (fibroblast), are plated at 3 x 104/well.2. Incubate at 37°C overnight.

Day 23. Dilution of the two components (antibody + protein) are prepared. The protein con-

centration used = 50 ng/mL.4. The antibody is prepared in “excess” (relative to the protein concentration, usually

just slightly lower than the ED50 or at the low end) concentrations of 1000X, 500X,200X, 100X, 10X and 5X.

5. In a fresh 96-well plate, 50 µL of the appropriate antibody dilution is added to 50 µLof the protein (duplicated for each antibody concentration).

6. Mix well and incubate 30 minutes at 37°C, 5% CO2.7. Following this, the plate containing the cells is removed from the incubator and the

media on the cells is carefully aspirated and discarded.8. The plate with the antibody + protein is removed and those solutions (100 µL) are

slowly and carefully added onto the cells. An additional 100 µL of fresh media plusActinomycin D is added and the plate returned to the incubator for 18 hours.

Results:For non-neutralization the monolayer of cells will be gone, i.e., the antibody did not blockTNF-α and therefore TNF-α destroyed the cells.

In general, once the antibody + protein incubation is complete, add your target cell andproceed as the standard bioassay conditions dictate (incubation time which allows forproliferation, etc.).

Always run as controls:•Protein alone + cells (e.g., if the neutralization doesn’t work very well, perform a

dose-response curve with the protein, then extrapolate from there which con-centration of protein to use for neutralization).

•Antibody alone + cells.•Cells + basal media.•Cells + complete media (mimic of the cells normal growth conditions).

Product Description Part Number SizePaxillin [pY31] 44-720 10 BlotPaxillin [pY118] 44-722 10 BlotPaxillin [pY181] 44-724 10 BlotPKR [pT451] 44-668 10 BlotPLCγ-1 [pY783] 44-696 10 BlotPro-Growth Sampler 44-586 7 mini-vialsPyk2/CAKβ/FAK2 [pY402] 44-618 10 BlotPyk2/CAKβ/FAK2 [pY579] 44-632 10 BlotPyk2/CAKβ/FAK2 [pY580] 44-634 10 BlotPyk2/CAKβ/FAK2 [pY881] 44-620 10 BlotPyk2/CAKβ/FAK2 [pYpY579/580] 44-636 10 BlotPyk2 Sampler 44-638 4 mini-vialsRb [pT356] 44-578 10 BlotRb [pT821] 44-582 10 BlotRb [pT826] 44-576 10 BlotRb [pSpT249/252] 44-584 10 BlotRb [pSpS807/811] 44-580 10 BlotRON [pYpY1215/1216] (mouse)

[pYpY1238/1239] (human) 44-442 10 BlotSrc [pY215] 44-658 10 BlotSrc [pY418] 44-660 10 BlotSrc [pY529] 44-662 10 BlotSrc Sampler 44-670 4 mini-vialsSTAT1 (human) [pY701] 44-376 10 BlotSTAT1 (mouse) [pY701] 44-378 10 BlotSTAT1 [pS727] 44-382 10 BlotSTAT3 [pS727] 44-384 10 BlotSTAT3 [pY705] 44-380 10 BlotStress Sampler 44-646 6 mini-vialsSyndecan-4 [pS179] 44-570 10 BlotTau [pT181] (human) 44-732 10 BlotTau [pT181] (mouse) 44-766 10 BlotTau [pS199] 44-734 10 BlotTau [pS202] 44-736 10 BlotTau [pSpS199/202] 44-768 10 BlotTau [pT205] 44-738 10 BlotTau [pT212] 44-740 10 BlotTau [pS214] 44-742 10 BlotTau [pT217] 44-744 10 BlotTau [pT231] 44-746 10 BlotTau [pS262] 44-750 10 BlotTau [pS396] 44-752 10 BlotTau [pS404] 44-758 10 BlotTau [pS409] 44-760 10 BlotTau [pS422] 44-764 10 BlotTau Sampler 44-780 13 mini-vials

2. ELISA Troubleshooting GuideAppendix

1. Antibody-Peptide Competition Experiments

The following protocol is used to demonstrate the specificity of an antibody by peptidecompetition. We have successfully used this protocol for a large number of affinity puri-fied antibodies that were raised against a peptide immunogen. The protocol can be usedto differentiate between phosphopeptides and non-phosphopeptides, as well comparingreactivity between similar regions of two proteins. It should be noted that anti-peptideantibodies don't always show differentiation between similar proteins although they mayindeed exhibit the appropriate specificity with the target protein. This observation stemsfrom the fact that peptides in solution may be much more flexible than the same peptidesequence in the context of the intact protein, where one or both termini are constrained.For this reason, a negative result in a peptide competition experiment cannot be used asa definitive proof of inappropriate reactivity.

1. Thaw antibodies on ice, and let lyophilized peptides come to room temperatureinside a desiccator.

2. Make antibody solutions in Antibody Sample Buffer: Set up 6 tubes in order totest 2 antibody concentrations and 3 peptide concentrations (0, 200-, and 500-fold molar excess). Mark 3 tubes as 0.25 µg/mL and 3 tubes as 0.75 µg/mL, indi-cating the final antibody concentration. Add a volume of antibody to samplebuffer required to yield a final antibody concentration of either 0.25 µg/mL or0.75 µg/mL in a final volume of 2 mL. Each solution will be used for it's owncompetition reaction.

3. Weigh peptides on the microbalance into a plastic (not glass) tube: 50 µg each.4. Reconstitute each peptide by adding 500 µL of ultra pure water (0.1 µg/µL [stock])

at room temperature. After peptide has dissolved, triturate several times using apipette. Do not introduce air bubbles if at all possible. The pH of solution shouldbe approximately 5.5 to prevent cysteine disulfide bonds from occurring.Therefore, you may need to add a drop of acetic acid to the peptide solution.

5. Fold the amount of stock peptide indicated in the table below into each antibodysolution and rock gently at room temperature for 30 minutes.

Table 14:

6. The rest of the peptide solution can be stored at -20°C in 20 µL aliquots.7. Prepare the blot and add 350-400 µL of this primary antibody-peptide mix to the

correct slot-blotter lane. Continue with the Western blotting protocol as usual.

Elevated BackgroundProblem: Insufficient washing and/or draining of wells after washing. Solution contain-ing either biotin or SAV-HRP can elevate the background if residual is left in the well.Solution: Wash according to the protocol. At the end of each washing step, invertplate on absorbent tissue on countertop and allow to completely drain and tap forceful-ly if necessary to remove residual.

Problem: Contamination of substrate solution with metal ions or oxidizing reagents, causingit to turn blue.Solution: Use distilled/deionized water for dilution of wash buffer and use plasticequipment. Use substrate which is clear and colorless.

Problem: Contamination of pipette, dispensing reservoir or substrate solution with SAV-HRP conjugate.Solution: Do not use chromogen that appears blue prior to dispensing onto the plate.Obtain new vial of chromogen. Use single-use, disposables for pipetting.

Problem: Improper dilution of SAV-HRP working dilution. SAV-HRP concentrate con-tains 50% glycerol, is viscous and therefore, difficult to accurately measure.Solution: Warm solution of SAV-HRP concentrate to room temperature, draw up slowlyand wipe tip with tissue to remove excess.

Problem: Chromogen exposed to light prior to use, resulting in a blue color.Solution: Keep chromogen in vial until ready to dispense into plate and then pour intoa reservoir to prevent contamination of the vial with equipment. DO NOT COVERplate with foil.

Problem: Incorrect incubation times and/or temperatures.Solution: Follow protocol (room temperature is 25 ± 2°C).

Problem: Evaporation of wells during incubations.Solution: Securely seal plate with plate cover during all incubations.

Problem: Improperly set up blanks.Solution: Follow protocol when designating blank wells. Blank wells contain onlychromogen and stop solution and results should be subtracted from the reading for allother wells.

Problem: Incorrect amounts of SAV-HRP or chromogen dispensed into wells.Solution: Follow protocol for volumes to dispense and check pipette calibration.

Antibody Peptide Added [µg/mL] (MolarConcentration to 2 mL in µL excess)0.25 µg/mL 0 µL [0 µg/mL] (0)0.25 µg/mL 20 µL [1.0 µg/mL] (200)0.25 µg/mL 50 µL [2.5 µg/mL] (200)0.75 µg/mL 0 µL [0 µg/mL] (0)0.75 µg/mL 60 µL [3.0 µg/mL] (500)0.75 µg/mL 150 µL [7.5 µg/mL] (500)

Problem: Errors in pipetting the standard or subsequent steps.Solution: Always dispense into wells quickly and in the same order. Do not touch thepipette tip on the individual microwells when dispensing. Use calibrated pipettes andthe appropriate tips for that device.

Problem: Reagents (lyophilized standard, coating antibody, etc.) from different kits,either different cytokine or different lot number, were substituted.Solution: NEVER substitute any components from another kit.

Weak/No Color DevelopsProblem: Reagents not at RT (25 ± 2°C) at start of assay.Solution: Allow ALL reagents to warm to RT prior to commencing assay.

Problem: Incorrect storage of components, e.g., not stored at +2-8°C.Solution: Store all components exactly as directed in protocol and on labels.

Problem: Incubation of SAV-HRP step at 37°C when RT is directed.Solution: Follow instructions for SAV-HRP step incubation temperature given in theprotocol.

Problem: Omission of any incubation steps.Solution: Follow incubation outlined in the protocol.

Problem: Standards diluted in serum, culture medium, or other solution.Solution: Standards must be diluted in the standard diluent/assay buffer as recommend-ed in the protocol.

Problem: Microwells were allowed to dry out.Solution: Do not allow microwells to sit uncovered. After loading, place adhesive stripover plate to protect from evaporation. Unused microwells should be discarded.

Problem: Incorrect chromogen/stop solution used.Solution: Use only the chromogen and stop solution recommended in the protocol.

Problem: Excessively cool laboratory temperature, i.e., < 22°C.Solution: Adjust laboratory temperature to 25 ± 2°C.

Problem: Reagents have expired.Solution: Check expiration dates upon receipt and use prior to expiration.

Problem: Wells have been scratched with pipette tip or washing tips.Solution: Use caution when dispensing into and aspirating into and out of microwells.

Problem: Plate read at incorrect wavelength.Solution: The correct wavelength is 450 nm.

Elevated Sample/Standard ODsProblem: Standards diluted in serum, culture supernatant or other.Solution: Dilute standards ONLY in standard diluent/assay buffer as recommended inthe protocol unless another matrix is validated.

Problem: Incorrect dilution of the SAV-HRP conjugate.Solution: Warm solution of SAV-HRP concentrate to room temperature, draw up slowlyand wipe tip with kim-wipe to remove excess. Dilute in the standard diluent/assaybuffer or as directed by manufacturer.

Problem: Plate covered with foil during chromogen step.Solution: Do not cover plate with foil. Place plate in a drawer or some other light-freechamber during last incubation step.

Problem: Incubation times extended.Solution: Follow incubation times outlined in protocol.

Problem: Incubations carried out at 37°C when RT is recommended.Solution: Perform incubations at RT = 25 ± 2°C when instructed in the protocol.

Problem: Contamination of pipette, dispensing reservoir or substrate solution with SAV-HRP conjugate.Solution: Do not use chromogen that appears blue prior to dispensing onto the plate.Obtain new vial of chromogen.

Problem: Chromogen exposed to light prior to use.Solution: Exposure of the chromogen to light causes the solution to change from clearto blue. Keep chromogen in vial until ready to dispense into plate and then pour into areservoir to prevent contamination of the vial with equipment. DO NOT COVER theplate with foil.

Poor Standard CurveProblem: Improper preparation of standard stock solution.Solution: Dilute lyophilized standard as directed by the vial label only with the stan-dard diluent/assay buffer.

Problem: Standard was not diluted with the standard diluent/assay buffer.Solution: Standard should be diluted with the standard diluent/assay buffer unlessanother matrix is validated.

Problem: Inadequate washing and draining of wells.Solution: Wash according to the protocol. At the end of each washing step, invertplate on absorbent tissue on countertop and allow to completely drain and tap forceful-ly if necessary to remove residual.

3. Guidelines for the Development of Western Blotting Methods

Extracts: These should to be made using a buffer containing a panel of protease andphosphatase (especially for phospho-specific pAbs) inhibitors. They should be preparedon ice and centrifuged at 14,000 x g following solubilization or homogenization to clar-ify. The resulting supernatant should be aliquoted, rapidly frozen and stored at -80°C.Thawing should be carried out slowly on ice and the unused extract discarded.Typically 10-30 µg of extract protein is used for a mini-gel, using more will generallycreate more problems with backgrounds.

Blocking agent/Membrane: BSA (1-5%) with nitrocellulose is the best combination.Nitrocellulose is somewhat harder to work with but is less prone to background. If aprotein binds poorly to nitrocellulose, then other membranes should be tried (such asnylon-based versions such as PVDF or Immobilon). Pore sizes of 0.45 µm tend to workbetter than 0.22 µm. With PVDF or Immobilon, 4% BSA is generally the best blockingagent. But with nitrocellulose, both 4% BSA or 5% Skimmed Milk should be tried toblock. With most antibodies, BSA is better, but we find that with our JAK/STAT anti-bodies milk is better.

Washing: Using multiple washes +/- Tween 20 (0.05%) for up to 15 min each can alsomake a dramatic difference.

Antibodies: With both the primary and secondary antibodies, one needs to perform aseries of titrations to assure that the antibodies are at or near saturation, as opposed to10-100-fold above saturation (another common mistake) which can result in high back-ground and/or cross-reactivity with related forms of the protein. For secondary anti-bodies, we find that the Goat F(ab’)2 anti-species IgGs conjugated to either HRP orAlkaline Phosphatase work better than whole molecule IgGs and result in lower back-ground and minimum cross-reactivity. A 1:10,000 dilution of the secondary antibodyis an acceptable starting titer to determine the optimal final concentration. Other com-mercially available secondaries often give high/variable background that can be seenby running the Western with everything but the primary antibody, which is a valuablecontrol. Running the secondary alone without the primary is a valuable control to assessthe background of the antibody and helps titer it out to reduce non-specific binding.

Detection: The Tropix Western Star kit with Kodak’s BioMax™ (ultraclear) film appearsto be the best combination. This chemiluminescent reagent provides saturating kinet-ics as opposed to the flash kinetics seen with ECL and it tends to provide a less diffusebanding pattern. It also offers the advantage that the signal does not fade as rapidly asECL and the membrane can be exposed up to 24 hours later and produce the same sig-nal. The Western conditions should be adjusted to get a good signal in 30-60 seconds.

Problem: Incorrect dilution of the SAV-HRP conjugate.Solution: Warm solution of SAV-HRP concentrate to room temperature, draw up slowlyand wipe tip with tissue to remove excess. Dilute in the standard diluent/assay bufferor as directed by the manufacturer.

Problem: Inadequate collection system for analyte being measured.Solution: Some analytes require special handling to make accurate measurements, e.g.,collection of blood in heparinized vacutainer tubes will hinder accurate measurementof TNF alpha.

Problem: Attempt to measure analyte in a matrix for which the paired antibodies havenot been optimized.Solution: Please contact BioSource Technical Service for advice when using non-vali-dated sample types.

Poor PrecisionProblem: Errors in pipetting the standards, samples or subsequent steps.Solution: Always dispense into wells quickly and in the same order. Do not touch thepipette tip on the individual microwells when dispensing. Use calibrated pipettes andthe appropriate tips for that device.

Problem: Wells have been scratched with pipette tip or washing tips.Solution: Use caution when dispensing and aspirating into and out of microwells.

Problem: Liquid has been transferred from well to well during incubations.Solution: Do not use an orbital shaker during incubations. Peel adhesive plate coveroff carefully.

Problem: Incorrect volumes of materials dispensed into microwells.Solution: Follow protocol for reagent dispensing volumes. Check calibration ofpipettes.

Problem: Standard was diluted with the serum, culture medium or other buffer.Solution: Standard should be diluted with the standard diluent/assay buffer recom-mended unless another matrix is validated.

Problem: Particulates or precipitates found in the samples.Solution: Remove any particulates/ precipitates by centrifugation prior to dispensinginto the assay.

Problem: Dirty microwells-visible debris within or on bottom of microwells.Solution: Inspect microwells and invert plate to remove debris. Wipe bottom of plate with an absorbent tissue after each wash step. Never insert tissue into microwells.

Problem: "Edge effect"-due to uneven temperature from the outer edge wells to the wells in the center of the plate.Solution: Seal the plate completely with cover during incubations and place plate incenter of incubator when 37°C incubation is indicated.

4. Suggested Vendors96-well polystyrene microtiter plates

• Dynex Immulon 2HB, Catalog #6506• Nunc Maxisorp, Catalog #468667

Lysing Solution for FACS• Becton Dickinson, Catalog #349202

Avidin-Biotin-Peroxidase• Vectastain, Vector Labs, Catalog #PK-4000

DAB• Sigma, Catalog #D5905

Mounting Medium• GelMount Fisher, Catalog #BM-M01• Vectashield,Vector Labs, Catalog #H-1000

Sorbitol• Sigma, Catalog #S-3889

Anisomycin• Sigma, Catalog #A-9789

Protein Assay Kit• BioRad, Catalog #500-0006

Protein A Sepharose•Pharmacia, Catalog #17-0469-01

Protein A/G beads• Pierce, Catalog #20421 or #20422

Developing Solutions• BioRad, Catalog #170-6539, #170-6532• Tropix, Catalog #MS250R

PVDF Membrane• Fisher, Catalog #PV4HY00010

Tris-Glycine precast gels• Novex, Catalog #EC6029 or #EC6028

Western Blue• Promega, Catalog #W3960

Nitrocellulose Membrane• Novex, Catalog #LC2001

Milk• BioRad, Catalog #170-6404

ECL• Amersham, Catalog #21060L/93/01

Autoradiography film• Kodak, Catalog #876-1520

Once the optimum conditions are established, experiments can be carried out with a highdegree of confidence. It is at this point that we systematically vary the protocol to purifya given antibody so as to optimize its performance in the assay. In this way we can alsosignificantly improve the overall quality of the assay.

References

ELISA:1. ELISA, Theory and Practice, John Crowther, (1995) Humana Press. Chapters 2, 3, 4, and 5.2. Sponholtz, D.K., (1995) Immunoassays in microplate wells: Optimization of binding by pas-

sive adsorption. IVD Technology, March.3. Douglas, A.S. and Monteith, C.A., (1994) Improvements to immunoassays by use of covalent

binding assay plates. Clin. Chem. 40:1833-1837. 4. Stevens, P.W. and Kelso, D.M., (1995) Estimation of the Protein-Binding Capacity of

Microplate wells using sequential ELISAs. J Immunol Methods 178:59-70.5. Stevens, P.W., et al., (1995) Assessment of adsorption and adherence of proteins to polystyrene

microwells by sequential ELISA analysis. Anal Biochem 225:197-205.6. Suter, M. and Butler, J.E., (1986) The immunochemistry of sandwich ELISAs. II. A novel system

prevents denaturation of capture antibodies. Immunol Lett 13:313-316.7. Kenna, J.G., et al., (1985) Methods for reducing non-specific antibody binding in enzyme-

linked immunosorbent assays. J Immunol Methods 85:409-419.

Flow Cytometry:1. Hermanson, (1995) In Bioconjugation Techniques by Academic Press, pp 303-305.2. Prussin, C. and Metcalfe, D.D., (1995) Detection of intracellular cytokine using cytometry

and directly conjugated anti-cytokine antibodies. J. Immunol. Methods 188:117-128.3. Longobardi-Givan, A., (1992) In Flow Cytometry, First Principles by Wiley-Liss, pp. 75-102.4. Leukocyte Antigen Fact Book, A.N. Barclay, et al., (1997) Academic Press, pp.21-22.

I/PWestern:1. Boulton,T.G., et al., (1991) Purification and properties of extracellular signal regulated kinase 1,

an insulin-stimlated microtubule-associated protein 2 kinase. Biochemistry 30:278-286.2. Lange-Carter, C.A., et al., (1993) A divergence in teh MAP kinase regulatory network defined

by MEK kinase and Raf. Science 260:315-319.3. Burnette, W.N., (1981) “Western Blotting:” electorphoretic transfer of proteins from sodium

dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detectionwith antibody and radioiodinated protein A. Anal Biochem 112:195-203.

4. Laemmli, V.K., (1970) Cleavage of structural proteins during the assembly of the head of bac-teriophage T4. Nature 227:680-685.

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