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PROTOCOLS CELL COMMUNICATIONS LABORATORY
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Protocols - Cell Communications Laboratory
protocols
Cell communications laboratory
Edited by Virginia Monardes. July, 2003
Protocols Index
3A: ANTIBODIES
21B: APOPTOSIS
32C: BIOCHEMISTRY
106D: LIPIDS BIOCHEMISTRY
143E: CELL BIOLOGY
181F: CELL CULTURE
206G: PROTEIN FUSION
217H: MOLECULAR BIOLOGY
A: ANTIBODIES4WESTERN BLOTS
6PURIFICATION OF IG FROM SERA USING SEPHAROSE-PROTEIN A
8ELISA
ELISA PROTOCOL910ELISA PROTOCOL AQ
11FACS PREPARATIONS
12IMMUNOFLUORESCENCE
14COUPLING PEPTIDES TO CARRIER PROTEINS
15MOUSE MONOCLONALE ANTIBODY AFTER PEPTIDE INJECTION
16IMMUNOPRECIPITATION WITH T-CELL EXTRACTS
17OVERLAY ASSAY
18OVERLAY ASSAY WITH PKC
20ANTIBODY PURIFICATION FROM HYBRIDOMA SUPERNATANTS
WESTERN BLOTS
November 1991
ALWAYS WEAR GLOVES
Make transfer buffer and place in freezer to chill.
Nitrocellulose preparation:
Remove roll and gently expose membrane -- it is very brittle when dry.
Cut a piece the size needed using a curved-bladed scalpel or a razor (For a minigel 2.5 in x 3.5 in)
Handle with flat surfaced forceps
Lay the cut nitrocellulose on buffer in a pan, first lay it on the surface and then allow it it slip under the buffer. You may stack several pieces in the buffer.
Pad and sponge preparation:
Soak the pads (3M paper or Biorad transfer pads) and sponges in transfer buffer. This buffer may be saved and reused many times for this purpose. It is critical that there be no bubbles trapped in the pads and sponges.
Set up a stirrer and cover with paper to soak up any spills.
Place the tank with the transfer chamber on the stirrer, add a stirbar and 800 ml ice cold transfer buffer. Make sure the stirbar spins freely. Add white plastic ice insert to the back of the chamber. Orient the tank so that the ice is in back, and the red pole of the transfer chamber is to your right. This means the black side of the transfer chamber is to the back.
To prepare the sandwich:
1.) Transfer the soaked pads to the buffer tray with the open holder. Squeeze out all bubbles. With the holder open so that the black side is down under the buffer and the clear side is nearest you, place a sponge topped with a 3M pad on the black back.
2.) Prepare the gel by a) removing the stacker - get rid of all of it because it will stick to the nitrocellulose and make a huge mess. b) mark the lower right hand corner of the gel for orientation, then c) using a spacer lift the gel and place it in the same orientation on the lower end of the pad (this is so that when the sandwich is turned upright and buffer drains out no air bubbles will be introduced around the gel and prevent transfer.
3.) Using membrane forceps place the nitrocellulose directly over the gel, if measured correctly it should fit well with space on all sides. No bubbles!
4.) Place another 3M pad on top of the nitrocellulose NO bubbles! followed by another sponge. Align all the pads so they do not stick out the edges of the holder (if they do the sandwich will not fit into the transfer apparatus)
Place the first sandwich in the transfer holder with the black side of the holder next to the black panel of the apparatus and prepare the second sandwich if it is to be used.
Run at 30 V overnight for medium to largish proteins, less time for peptides, more for giant proteins. It may also be run at up to 200 V for 1-2 hours.
To remove the blot from the holder:
1.) Turn off the current
2.) Open up the sandwich so the nitrocellulose is on top of the gel (black side down, open the clamp towards you)
3.) Mark the corners of the gel on the nitrocellulose so you can see where the gel was. This will be on the back of the blot. The prestained size markers will prove that the transfer occurred.
4.) Transfer the nitrocellulose to a bath with blocker (usually 3% non-fat milk powder in PBS with 0.02% NaN3.) Block for at least 2-4 hrs, overnight is better. Make sure there is plenty of blocker so that the membrane is floating freely and the shaker is on highish speed. (blocking solution may be reused.)
5.) Rinse briefly with PBS
6.) Incubate with first antibody 10 ml at whatever dilution is appropriate x 2 hr
7.) Wash 3 x 5-10 min with PBS
8.) Incubate with second antibody (Biorad horseradish peroxidase, 1:3000) x 2 hr
9.) Wash 10min with PBS
10.)Wash 10 min with 100 ml PBS with 2 drops Tween 20
11.)Wash 10 min with PBS
12.)Add developer made fresh just before using. If the blot has not developed within 10 min you may add another 10 l H202. Rinse extensively with water. Membranes may be stored dry wrapped in a Kimwipe and foil.
PURIFICATION OF IG FROM SERA USING SEPHAROSE-PROTEIN A
Material
Saturated (NH4)2SO4 : at 25oC saturation for 761 g/l (4.1 M)
Tris Buffer pH 8.5 0.1 M Tris12.12 g
0.150 M NaCl 8.77 g
2.5 mM NaN3 0.16 g
H2O to 1 liter
(for frequent experiments make a 10x concentred Buffer)
Dialysis tubing : Spectrapor 4, M.W. Cutoff 12-14.000
10x PBS pH 7.0-7.5
Na2HPO4 2H2O 89 g
KHPO413.6 g
NaCl58 g
H2Oto 1 liter
Glycine 0.1 M-HCl Buffer pH 3
Protein A Sepharose CL-4B (Pharmacia No. 17-0780-01)
Tris-HCl pH 8.81.5 M Tris
Tris-HCl pH 6.80.5 M Tris
30% acrylamide/bisacrylamide (38 : 2)
20% SDS
TEMED
10% APS
a) (NH4)2SO4 precipitation of Ig from seraWhile the serum is being stirred gently on ice with a magnetic bar, add sarurated ammonium sulfate dropwise to bring a final concentration of 50 %. Alow sufficient time for precipitation by stirring for 6 hr or overnight at 4 C.
Centrifuge at 8000 RPM (3000x g) in the Kontron (rotor A 8.24) for
30 min.
Carefully remove and save the supernatent for gel analysis. The pellet is resuspended in a minimum volume of Tris Buffer pH 8.5 (50 ml Tris for every 20 ml of starting serum).
Mesure OD 280 before dialysis.
b) Dialysis of resuspended proteins after ammonium sulfate fractionationDialyse 3x times in 10 volumes of Tris Buffer pH 8.5 for 2 hr, last time overnight at 4oC.
Mesure OD 280 after dialysis.
c) Affinity chromatography on sepharose-protein A columnSwell the beads in 1x PBS and pull them in an appropriate column.
1.5 g of dry beads gives 5 ml of swollen gel and can fixe 100 mg of pure Ig.
Wash the column with 20 volumes of Tris Buffer pH 8.5.
When all the Buffer has passed, gently charged the dialysed suspended proteins on the column. Collect 3ml fractions with a slow debit until all the suspention has penetrated the gel. Then eluate the non interacting proteins with Tris Buffer pH 8.5 with a fast debit, collecting 3ml fractions and measuring OD 280.
When OD 280 is down to baseline, eluate the fixed Ig with the Glycine buffer pH 3. Collect 3ml fractions into tubes already containing 1.5 ml of Tris Buffer pH 8.5 to immediately neutralise the acidic pH of the elution buffer. Monitor OD 280 to check when elution is complete.
Regenerate the column by passing 10 volumes of Tris Buffer pH 8.5, 10 volumes of NaAc-NaCl Buffer pH 4.5 and then again 10 volume of Tris Buffer pH 8.5 through the column.
Store the column at 4oC in Tris Buffer pH 8.5 in the presence of 0.3 % NaN3.
d) Mini SDS-PAGE analysis of the Ig purification
Check purification by SDS-PAGE analysison a 10 % mini gel.
Load 1-5 g per lane of the proteins before purification, the non-interacting proteins in flow through and wash fractions and the purified proteins.
Run the purified Ig fraction under both reducing and non-reducing conditions and compare with a standard IgG fraction containing both the heavy and light chain (e.g Bio Rad G_R IgG (H+L) ).
Also add a molecular weight marker.
ELISA
1) Let dry 10 l of antigen per well, placing the plates in dessicator overnight.
For pure Ag use approx. 10 ng/well.
2) Rinse the plate with PBS and saturate free sites with PBS-1% BSA-0.05% Tween 20. Incubate 1 hr at 37oC.
3) Rinse again with PBS and keep the plate moiturized until use. Store at 4oC.
4) First row of the plate is used as a blank so, it has to be incubated with 50 l/well of the solution in where the antibodies are (control medium). Add 50 l of the first antibody (hybridoma supernatant) and incubate for 1 hr at 37oC.
5) Wash the plate with PBS-0.05% Tween 20. 5 x 5 min.
6) Add 50 l of the second antibody diluted in blocking solution (a-mouse IgG-HRP diluted 1:500). Incubate 1 hr at 37oC.
7) Wash as indicated in #5.
8) Incubate with 100 l of substrate solution (15-60 min, room temperature). Prepare solution immediately before use.
0.504g Citric acid
1.367g Na2HPO4.7H2O
100 ml MQ-water, pH 5.0-5.2
Per 10 ml of this buffer add 0.02g o-phenylenediamine.2HCl (OPD) (light sensitive and mutagenic), and immediately before use add 6.7 l of 30% H2O2.
9) Read in ELISA-reader at 450 nm.
ELISA PROTOCOL
(after the protocol used in N. Fasel's Lab, 1995)
1.Coat each well to be used on plate with 100 l antigen solution (take 100 ng to 10 g protein or peptide per well; 0.5 g of peptide seems to work well).
2.Wrap box and leave overnight at 4 C.
3.Block unspecific binding by addition of 100 l 2x blocking solution per well, incubate for two hours at room temperature.Meanwhile the dilution of the serum/lysate to be tested in antibody dilution solution can be prepared and left at room temp. for 30 min.
4.Remove blocking solution by virtually "throwing" liquid content away and remove remaining drops by banging uspide-down against the bench sufrace covered with paper towel. Wash four times with 100 l of 1x PBS.
5.Put 100 l of the preincubated antibody/lysate-mix into each well and incubate for 1 hr at RT. (Dilution series: Put 100 l of antibody dilution buffer into each well; add to well supposed to contain the highest concentration 100 l of the 1:10 diluted serum, mix well. Take 100 l from this well and add to the next well, mix, take 100 l and so on).
6.Discard the solution and wash wells four times with 1x PBS, discard each time.
7.Put 100 l of the diluted 2nd antibody solution into each well and leave 1hr at RT.
8.Wash four times with 100 l PBS, discard each time.
9.Put 100 l of NPP-containing developer solution into each well and leave at RT until yellow colours appears (10 min.-60 min).
10. Measure absorption at 405 nm.
Solutions/Material:
1x PBS
used to dilute antigen coating solution
2x blocking solution1xPBS, 5% milk powder, 0.05% Tween20
antibody dilution 1xPBS, 2.5% milk powder, 0.05% Tween20 solution
2nd antibody
Polyclonal anti mouse (anti rabbit etc.) IgG coupled to alkaline phosphatase (SIGMA, dil 1:1000)
Developer solution1M Diethanolamin (pH=9.8), 1 mM MgCl2 (before
development Nitrophenyl-phosphate pills are added
to a final conc. of 1 mg/ml)
Plates: NUNC ImmunoPlates, MaxiSorp F96
ELISA PROTOCOL AQ
Protocol for ELISA detection of proteins and peptides.
Incubate plates with 100ul of 10ug/ml antigen solution (either ovalbumin or hemocyanin) for about 1 hour. With peptides use about 100-200 ng per well. Incubate overnight at 37C. Leave wells of the first column balnk.
Wash plates with PBS, then block for 1 hour at 37C with PBS containing 1% BSA and 0.05% Tween 20. Use 200ul per well.
Rinse again with PBS and keep plate moisturized until use. Store at 4C.
The first column of the plate is always used as a blank, so nothing is coated there and it is incubated with 100ul per well of the solution in which antibodies are diluted. Each row is incubated with the appropriate antibodies diluted as desired. To test anti-sera, serial dilutions in steps of 2 starting with a dilution 1:10 generally works well. Incubate 1 hour at 37C.
Wash the plate with PBS containing 0.05% Tween 20, 5 x 5 min.
Add 100 ul second antibody diluted in blocking buffer. Incubate 1 hour at 37C.
Wash as above.
Incubate with 100ul of substrate solution. Prepare solution immediately before use:
0.504g citric acid
1.367g Na2HPO4.7H2O
100 ml Ultrapure water, pH 5.0-5.5
Per 10ml of this buffer add 0.02g o-phenylenediamine.2HCl (OPD) (light sensitive, mutagenic) and immediately before use add 6.7ul of 30% H2O2.
Read in Bio-Rad ELISA reader at 450 nm at various time intervals.
Comments:
Best second antibodies to rabbit (H+L chains) from Kirkgaard & Perry. Others from Bio-Rad also work. Protocol can be modified to detect protein-protein interactions (see Ghosh et al., 1994)
AQ, 2-8-97
FACS PREPARATIONS
A) Intracellular staining
Solutions
Sol A : 1xPBS/2.5% FCS/0.05% NaN3
Sol B 10x : 10x PBS/0.02% NaN3/1% saponin
Sol B 1x
1e antibody : diluted in Sol A
Take 9 V antibody + 1 V sol B 10x
Amount needed : 100 l for each reactions
Test different concentrations (typically a serum must be diluted 100-1000x)
2e antibody : diluted as fallowed
Suspend 2 mg antibody (FITC or DTAF conjugated, Jackson ImmunoResearch) in 1.5 ml steril H2O. Leave for 1-2 hours at RT on a rotative shaker protected from light. Stock aliquots at -20 C.
For one FACS experiment :Antibody5_
Sol. B 10x100_
Sol. A 895_
Method
1.Count 105-106 cells for each experiment.
2.Wash them 2 times with PBS.
3.Resuspend the cells in 1 ml 4% paraformaldehyde + 10 l Sol. B 10x and fixe them for 10 minutes at RT.
4.Wash 2 times with PBS.
5.Add 100 l of first antibody for each experiment and incubate for 30 minutes at RT.
6.Wash 3 times with sol. B.
7.Add 100 l of second antibody for each experiment and incubate for 30 minutes at RT.
8.Wash 3 times with sol. B.
9.Suspend the cells in 100-300 l PBS for FACS.
10.If cells have to be keep more than 3 hours fixe them for 10 minutes in 1 ml 4% paraformaldehyde. Then wash them 3 times with PBS and suspend them in 100-300 l of sol. A for FACS.
IMMUNOFLUORESCENCE
Preparation of Swiss 3T3 fibroblasts: adherent cells. Cells grown on coverslips.
Coverslips (Microscope Cover glasses, 14 mm;
Coverslips are stored in 70% ethanol at 4C. They are flamed and put in a 10 cm Nunc dish.
Cells are allowed overgrow the cover slips and stimulated as described in the "cell culture" protocol.
Remove the medium (with or without stimulator).
Fixation: Incubate with paraformaldehyde 4% for 30 minutes at RT. Be careful, paraformaldehyde is highly toxic.
Wash 5 x 3 minutes with PBS at RT.
Permeabilization: 10 minutes with 0.2% Triton X-100 in PBS at RT.
Wash 5 x3 minutes with PBS at RT.
Block for 30 minutes at 37C with 4% BSA (Sigma; A-2153) in PBS.
First antibody:- Overnight at 4Cor:
- 3 hours at RT
Put a piece of parafilm in a box containing humid paper and put 10 l of antibody solution on parafilm. Put the coverslips on drops.
Dilution for all antibodies:1:50 in blocking solution.
- mouse IgM anti-caveolin-1 (monoclonal; Transduction Laboratories, C13620)
- rabbit anti-PKC (polyclonal; Sigma: P4334 (PKC), P8333 (PKC), P8458 (PKC) and P0713 (PKC).
Wash 5 x 3 minutes with PBS / 0.01% Tween (Sigma, P-1379) at RT.
Secondary antibody:1 hour at 37C protected from light.
Put 10 l of antibody solution on parafilm in a humidified box.
Dilution: 1:400 in blocking solution
- anti-mouse-Cy3 (Jackson, 115-165-144)
- anti rabbit- FITC (Jackson, 111-095-144)
Wash 5 x 3 minutes with PBS / 0.01% Tween at RT.
Rinse briefly in dH2O to remove salt.
Mount in FluorSaveTM Reagent ( Calbiochem, 345789) on glass slides which were previously cleaned with ethanol 70%.
Store at 4C protected from light.
Analysis by confocal microscopy (Zeiss).
Wavelength for FITC:488 nm
for Cy3:543 nm
Preparation of 4% paraformaldehyde:Warm PBS at 65C in water bath. Place everything in ventilated hood. 20 g of p-formaldehyde (Fisher) are added to the warm PBS and let stir for over 1 hour. It does not need pH adjustment, but check with pH paper that it is around 7. Add more PBS to complete volume to 500 ml and aliquot in 50 ml tubes.
Store at -20C for several months. Solution may be thawed several times.
SEQ CHAPTER \h \r 1
SEQ CHAPTER \h \r 1COUPLING PEPTIDES TO CARRIER PROTEINS
Protocol for activation of any protein (example: Limulus Polyphemus Hemocyanin) to permit subsequent reaction with free -SH groups present in cysteines of peptide to be coupled.
Make 1.5 mls of Limulus Polyphemus Hemocyanin (Sigma H1757) solution at 20mg/ml in 50mM NaPi buffer pH7
Put a 1 ml aliquot without creating foam into an Eppendorf tubes
Add 20ul MBS (Sigma M8759) dissolved at 25 mg/ml in dimethyl-formamide (Fluka, puriss).
Incubate protein for 1 hour at room temperature. Then load 1ml of solution onto a gel filtration column (Swift TM, Pierce) equilibrated in NaPi buffer, calibrated to permit separation of activated protein from MBS. Collect 1ml fractions and pool protein peak, about 4 ml volume (fractions 6-10). Re-run protein peak over column; collect again 1ml fractions; pool the 4 protein peak fractions as activated KLH (enough for 2-3 coupling reactions).
To an Eppendorf tube containing 7.5 mg of peptide add 1.5 mls of activated LPH solution (final peptide conc. about 2.5 mM). Leave standing at room temperature for 30 min., then put on rotating shaker overnight. In most cases a precipitate will form immediately. Sometimes the peptide will dissolve, then slowly turn opalescent and precipitation will occur later on.
Next day, the product of each coupling reaction is mixed with 3ml of PBS and split up into 5 portions of 0.9ml. One such portion is mixed 1:1 with Freunds complet or incomplete adjuvant for subcutaneous injection into rabbits.
Comments:
Same protocol has been applied to couple peptides to ovalbumin. The most crucial step is getting good separation of activated carrier protein and free MBS, otherwise the latter will prevent peptide coupling later on.
AQ, 2-8-97
MOUSE MONOCLONALE ANTIBODY AFTER PEPTIDE INJECTION
MaterialYoung BALB-C mice
Peptide diluted 2 mg/ml : MAP, reverse-MAP or free peptide
Adjuvant Freund Incomplete (AFI)
1 and 2 ml syringes : Becton Dickinson
Orange needles : Becton Dickinson 25G5/80.5x16
Box for injection (GP's lab)
MethodeMix peptid and AFI in a 1 : 1 ratio in parafilm sealed 2 ml syringe .
Sonicate the mix briefly 2-3 times until you get a nice emulsion.
Inject the content of the syringe into a 1 ml sirynge.
Eventually put the syringe vertically in the frige to allow air bubbles to go out.
Inject with an orange needle 50 l (50 g) of the emulsion (see below the scheme for injection). The emulsion provokes a swelling at the base of the tail.
SEQ CHAPTER \h \r 1IMMUNOPRECIPITATION WITH T-CELL EXTRACTS
The T-cell extracts (total volume 1ml) in Crumptons lysis buffer (CLB) containing 10 mM iodoacetamide and protease inhibitors (PMSF, 50uM final conc.) are first pretreated with Pansorbin in round-bottomed Falcon 2063 tubes for 2 hours at 4C, and then after removing the supernant (spin 10min., 2500 rpm) over night at 4C with a fresh aliquot of washed Pansorbin.
The supernatant is treated next day at RT for 1 hour with 50ul Protein A beads (Pharmacia # 17-0780-01). At this point the remaining supernatant is split into x batches (depending on number of IP exp.) corresponding to roughly 107/x initial T-cells.
To each volume of 500ml pre-treated cell extract 500ml CLB and 10ul or more of affinity purified IgGs are added and incubated over night at 4C. Depending on the affinity of the antibody, this incubation may be reduced to 2-3 hours.
Next day add 50ul ProtA beads and incubate for 1 hour at RT. Then remove supernatant and wash beads 6x with wash buffer in small Eppendorf tube at RT. Spin each time 1min. at 1000rpm in Eppendorf centrifuge.
The proteins specifically bound are eluted from the beads with sample buffer, 1min. at 100C, in the presence of 1M DTT and 2M iodoacetamide to alkylate thiol groups
The supernatant from the initial IP can be reused to immunoprecipitate another antigen, as long as it did not coprecipitate with the first. To remove residual ProtA beads this supernatant must be filtered though 0.2um filters (Schleicher & Schuell, FP030/3). Then the process is repeated, starting with the incubation with the antibody.
COMMENTS/OBSERVATIONS
AQ 17-5-95
SOLUTIONS
Crumptons Lysis Buffer:0.5% NP-40, 0.5% Deoxycholate, 50mM NaCl, 25mM Tris-HCl pH=8.2
Wash buffer:
0.5% NP-40, 500mM NaCl, 10mM EDTA, 125mM Tris-HCl pH 8.2.
PanSorbin
(Calbiochem. #507858)Place 2.5ml of 10% Pansorbin into a round-bottomed Falcon 2059 tube. Add 6ml CLB and mix. Spin at RT, 10min., 2500 rpm. Discard supernatant. Resuspend pellet in 6ml CLB, mix and repeat centrifugation. Resuspend pellet again in 6ml CLB, distribute 6x1ml aliquots into round bottomed F2063 Falcon tubes. Spin as above and store tubes at 4C until needed.
SEQ CHAPTER \h \r 1
SEQ CHAPTER \h \r 1
SEQ CHAPTER \h \r 1OVERLAY ASSAY
Run SDS-PAGE using Bio-Rad mini-gel system, loading max. 50-100 ug of protein per lane. For lung extracts, as little as 10-20 ug protein are required to get a signal in the overlay assay, for other tissues or extracts from cultured cells as much protein as possible should be loaded.
Transfer to nitrocellulose at 30V over night. Cool unit to 4 C on ice.
If necessary, cut blot into strips (stain region corresponding to gel top using Ponceau Red S or Amid Schwarz). Block at least for 24 hours in block solution at 4 C.
Wash for 15-30 min. in PBS.
Then incubate with GST-PKC in fusion protein dilution buffer at 10ug/ml for 2 hours in cold room at 4 C on tilting shaker.
Wash 3x 10 min. at room temperature on rotating shaker (Tschopps lab) in large volumes of PBS (AQs version)/0.04% NP-40.
Incubate with first antibody (1Ab), rabbit-anti-GST (serum#218) diluted 1:2000 in 1Ab dilution buffer, for 1.5 hours on rotating shaker (Tschopps lab).
Wash 3x 10 min. in PBS (AQs version) at room temperature on rotating shaker (Tschopps lab).
Incubate with 2Ab, Goat-anti-rabbit IgG HRPO coupled (Bio-Rad) diluted 1:3000 in 2Ab dilution buffer, for 1.5 hours on rotating shaker (Tschopps lab) at room temperature.
Wash 3x PBS (AQs version). Second wash should contain 0.04% NP-40.
Develop at room temperature with chloronaphtol/hydrogen peroxide in PBS, i.e. 9 ml PBS (AQs version), 1 ml chloronaphtol stock (stored at -20C), 10 ul H2O2 (30% conc., stored at 4C, light-protected).
Comments:
Results/Conclusions:
Solutions for Overlay assay:
AQs 10x PBS - 8.5g NaCl(Mw 58.4)/8.8g NaHPO4 (Mw 142)/1.9g NaH2PO4.H2O (Mw 138) in 1l final volume. pH of 1x PBS should be around 7.2.
Block solution - 3.5% milk powder in PBS (AQs version) with 2mM sodium azide.
GST-PKC fusion protein dilution buffer - To PBS with 0.04% NP-40 add 10% ethylene glycol.
1Ab dilution buffer - PBS with 10mg/ml BSA. Add 2mM sodium azide for antibody storage.
2Ab dilution buffer - PBS with 10mg/ml BSA. Add NP-40 to 0.04% final concentration. Do not add sodium azide.
Chloronaphtol stock - 30 mg of chloronaphtol (Peroxidase developer from Bio-Rad) per 10ml of ethanol.
AQ, 4-1-95
SEQ CHAPTER \h \r 1OVERLAY ASSAY WITH PKC
Run SDS-PAGE using Bio-Rad mini-gel system, loading max. 50-100 ug of protein per lane. For lung extracts, as little as 10-20 ug protein are required to get a signal in the overlay assay, for other tissues or extracts from cultured cells as much protein as possible should be loaded. For purified proteins 1-2 ug total protein are sufficient.
Transfer to nitrocellulose at 30V over night. Cool unit to 4 C on ice.
If necessary, cut blot into strips (stain region corresponding to gel top using Ponceau Red S or Amid Schwarz). Block at least for 24 hours in block solution at 4 C. Wash for 15-30 min. in PBS.
Then incubate with PKC (Threonine Sepharose fraction 19-20, ......) in phosphorylation buffer (+/- ATP, +/- PDBu) for 1.5-2 hours RT on tilting shaker lab F413. Make PKC solution as indicated below
Phosphorylation buffer- Stock Solutions:
3 % Triton X-100: Dilute Pierce Surfact-Amp (28314 G) 10% to 3% with deionized water. This is 43 mM. For 5ml of a 10x stock use 1500ul; dilute to final vol. 5ml with ddH2O.
Phosphatidyl serine: Stock is 20 mg/ml (31 mM), mol. wt. 820, dioleoyl phosphatidyl serine from Avanti Polar Lipids. For 5ml of a 10x stock at 10 mol%, dry down 696 ml and resuspend in the 5ml of Triton X-100 3%
Dioctanoyl glycerol (diC8): Stock is 25 mg/ml (73 mM), mol. wt. 344, from Avanti Polar Lipids. For a 2 mol% stock, dry down 60 ul of DiC8 and resuspend in 5 ml of above PS /TX100 mix. This solution will actually be only 1.2 mol% sn-1,2-DiC8, the rest is inactive sn-1,3-diC8 as determined by DGK assay.
Working lipid mix: Dry PS in a glass disposable tube to make a final concentration of 10 mol % in 5 ml 3% Triton X-100. Use PS stock to dissolve diC8 to a final concentration of 2 mol %. Mix well with a vortex and allow to sit in a 37oC bath for a few minutes, mix well again. Make 0.5ml aliquots and freeze at -20oC. Dilute each aliquot to a final volume of 5 ml for phosphorylation on blots. Final assay concentration will be 0.3% TX-100, 10mol% PS and 1.2 mol% sn-1,2-diC8.
PKC overlay experiment: To one such lipid aliquot (0.5ml), add the following reagents
0.5 ml 200 mM Tris, pH 7.5
0.5 ml 50 mM MgCl2
0.5 ml 2 mM CaCl2
1 ml Thr. Seph. PKC peak (prep. 1 brain)
2 ml ddH2O
After PKC incubation, wash 3x 10 min. at 4oC on rotating shaker (cold room) in large volumes of PBS(AQs version)/0.04% NP-40.
Incubate with first antibody (1Ab), rabbit-anti-PKCg (Boehringer Mannheim) diluted 1:500 in 1Ab (AQs version) dilution buffer, for 1.5 hours on tilting shaker (F413).
Wash 3x 10 min. in PBS (AQs version) at 4oC on rotating shaker (cold room), second wash with 0.04% NP-40.
Incubate with 2Ab, Goat-anti-rabbit IgG HRPO coupled (Bio-Rad) diluted 1:3000 in 2Ab dilution buffer (AQs version), for 1.5 hours on tilting shaker (F413) at room temperature.
Wash 3x PBS (AQs version) in cold room. Second wash should contain 0.04% NP-40.
Develop at room temperature with chloronaphtol/hydrogen peroxide in PBS, i.e. 9 ml PBS (AQs version), 1 ml chloronaphtol stock (stored at -20C), 10 ul H2O2 (30% conc., stored at 4C, light-protected).
NB. Also ECL can be used to develop overlay blots, but washes must be more stringent, at least 4x7 min. at each step where washing is required
Comments:
Solutions for Overlay assay:
AQs 10x PBS - 8.5g NaCl(Mw 58.4)/8.8g NaHPO4 (Mw 142)/1.9g NaH2PO4.H2O (Mw 138) in 1l final volume. pH of 1x PBS should be around 7.2.
Block solution - 3.5% milk powder in PBS (AQs version) with 2mM sodium azide.
GST-PKC fusion protein dilution buffer - To PBS with 0.04% NP-40 add 10% ethylene glycol.
1Ab dilution buffer - PBS with 10mg/ml BSA. Add 2mM sodium azide for antibody storage.
2Ab dilution buffer - PBS with 10mg/ml BSA. Add NP-40 to 0.04% final concentration. Do not add sodium azide.
Chloronaphtol stock - 30 mg of chloronaphtol (Peroxidase developer from Bio-Rad) per 10ml of ethanol.
AQ, 26-3-97 SEQ CHAPTER \h \r 1
SEQ CHAPTER \h \r 1
SEQ CHAPTER \h \r 1 SEQ CHAPTER \h \r 1ANTIBODY PURIFICATION FROM HYBRIDOMA SUPERNATANTS
Washing buffer: PBS 1x
Elution buffer: 0.1M Glycine pH 3.0/ 0.5M NaCl (1.88g Glycine + 25ml 5M NaCl for 250 ml)
Buffers need to be cold before starting
1) Equilibrate the column (2ml of anti-rat Agarose, Sigma) with 50 ml of PBS
2) Do ammonium sulfate cut of 500ml of supernatant and dialyze with PBS o.n. at 4C
2) Load the column with the dialyzed antibody. Recover the flow through.
3) Wash the column with 100ml of cold PBS.
4) Prepare tubes for elution: Add 200l of 1M Tris pH 7.4 per tube and labelled them
5) Elute the column with Elution buffer and collect fractions of 2 ml.
6) Mix the eluate with the Tris after every tube collection
7) Monitor for protein at OD=280nm and pool those fraction containing the antibody
The column should be washed immediately with 80ml of PBS and 20ml of PBS containing azide if is not needed the same day.
With the flow through repeat steps 2-7 until no more Ab binds to the column
8)Concentrate and dialyze the antibody using Centricon 30 (Amicon)
(Tube#) A 280nm
1--------------
7--------------
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3--------------
9--------------
4--------------
10--------------
5--------------
6--------------
B: APOPTOSIS22ANALYSIS OF APOPTOTIC CASPASE-3 (FLUOROMETRIC)
23ANALYSIS OF APOPTOTIC DNA FRAGMENTATION ON AGAROSE GEL
24DNA CONTENT ASSAYS BY FLOW CYTOMETRY
26IN SITU CASPASE-3/CASPASE-8 ASSAY BY FACS
27VIABILITY ASSAYS BY FACS
28ASSESSMENT OF CHROMATIN CONDENSATION AND MORPHOLOGICAL CHANGES
29QUANTIFICATION OF PHOSPHATIDYLSERINE EXPOSURE BY FACS
30DNA FRAGMENTATION ASSAY
31CASPASE-3 ACTIVITY ASSAY (CHROMOGENIC)
ANALYSIS OF APOPTOTIC CASPASE-3 (FLUOROMETRIC)
References
- M.P. Boldin et al. (1996) Cell 85, 803-815.
- H.R. Stennicke and G.S. Salvesen (1997) JBC 272(41), 25719-25723.
Materials and solutions
For this assay, a fluorometer (eg Fluoroskan II) and special black ELISA plates (96 wells) are needed.
Ac-DEVD-AMC, as a powder from Alexis, is dissolved in PBS to a final concentration of 10 mM (200x conc.) and stored in aliquots of 50-100 l at -20 C.
CHAPS buffer: 5 mM CHAPS detergent, 2 mM MgCl2, 150 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl (pH=7.4), 2 mM DTT.
The assay resides on the fact that caspase-3 recognizes specifically the tetrapeptide motif DEVD. In the assay, this tetrapeptide is coupled to amino-methylcoumarine (AMC), which uncleaved is only slightly fluorescent. Upon liberation by caspase-3 cleavage, AMC becomes highly fluorescent, and allows thus quantification of caspase-3 activity. I did measurements with the B lymphoma cells A20. Upon incubation with 100 ng/ml FasL, first caspase-3 activity was observable after about an hour.
Protocol for A20 cells and Fluoroskan II:
Per condition, 2 mio cells, preferentially in duplicate, are taken (1-3 mio should work). The cells are incubated either in 24 well plates at 37 C, 5% CO2 (especially for incubations longer than 3 h) or in 1.5 ml tubes in a 37 C waterbath (up to 3 h, with resuspension of cells every hour). Do not forget to switch on/prewarm fluorescence about 1 h before measurement!
Then the cells are harvested by centrifugation 3 min. at 3000 rpm at 4 C. The cells are washed once with ice-cold PBS and are then lyzed in 100 l CHAPS buffer supplemented with 200xDEVD-AMC solution to a final concentration of 50 M (100 M does also work). Incubation for 30 min. at 37 C. Then the samples are spun for 10 sec. at 13'000 rpm to get rid of the unlyzed material. The supernatant is pipeted into wells of a black ELISA plate. Try to pipet to leftmost wells (starting with A1) to minimize scanning time.
The prewarmed machine is set to the preset excitation and emission wave-lengths "1" (excitation 355 nm, emission 460 nm), the plate is put into position. Measurement is started by "Start" button. If reading is not stopped, results are printed automatically, otherwise you can print them by pushing "1". Results can be read between 30 min and 2 h. Be aware of the upper detection limit of the machine, so it is preferable to take measurements at different time points!
After usage rinse plate several times with normal and then with deionized water. Turn off the Fluoroskan.
M. Hunn/ Aug. 98
ANALYSIS OF APOPTOTIC DNA FRAGMENTATION ON AGAROSE GEL
The dissolution of the nuclear membrane is a rather late event observed during apoptosis. In this way, DNAse I, which normally resides in the endoplasmatic reticulum, gains access to the nuclear DNA, cleaves the DNA between the nucleosomes, and creates thus fragments with a length of 180 bp or multiples of 180 bp. When these fragments are analyzed on an agarose gel (2%), a typical ladder-like pattern is observed in many cases. This is a typical sign of apoptotic cell death.
This protocol is adapted to the B lymphoma cell line A20.Two plates containing 1 mio cells in 4 ml medium are prepared. To those plates 2 l of 100 g/ml FasL (final 50 ng/ml) is given. Then the cells are incubated for 4 h at 37 C, 5% CO2.
The cell are then harvested: Spin cells in three 1.5 ml tubes (1 min. at 5000 rpm). Resuspend the individual cell pellet in about 300 l of PBS and put in a single tube, spin cells. Then the cell pellet is resuspended in 100 l PBS, and cells are lyzed by addition of 100 l phenol/chloroforme/isoamylalcohol (25:24:1). Vortex. Centrifuge the cells for 30 sec at 13000 rpm. Transfer 50 l of the water phase to a new tube.
Of this crude DNA preparation, 17.5 l are mixed with 2 l buffer H (BM: for restriction digests) and 0.5 l RNAse A (500 g/ml). Incubation 30 min. at 37 C. Analyze the DNA on a 2% agarose gel.
M. Hunn/ Aug. 98 (acc. to P. Schneider, TP)
DNA CONTENT ASSAYS BY FLOW CYTOMETRY
Purpose of the test: Quantify hypodiploid cell populations using propidium iodide (PI).
Materials
-PI Stock solution 1mg/ml ( dangerous!- this compounds is carcinogenic and mutagenic)
-Filterd PBS and FBS fetal bovine serum.
- Isotonic buffer
-5 ml FACS tubes (Falcon N Cat 352052).
-Methanol PA cold (-20C)
Methods:
1. Seed cells in a 24- or 6-well plate (options determined by experiment) in order to obtain at least 2*105 cells for evaluation.
2. For adherent cells, put the medium of each well in 1.5 ml eppendorfs tubes. Then add 100l of trypsin (for 24 wells plate) over the cells and leave at 37C until cells detach.
Note: When cells have been starved, add 100ul of FBS to cell samples to neutralize trypsin and avoid cell damage.
3. Centrifuge the cells for 7 min at 3000 r.p.m.
4. The pellet is resuspended vigorously by hand and tubes are kept cold on ice.
5. The cells are resuspended in methanol 95% (-20C, 1ml for each 1 x 106 cells) adding the methanol drop by drop mixing gently with vortex.
1. Incubate the cells for 10 min at 20C. After this step, cells can be stored subsequently for one week at 4oC.
6. Centrifuge the cells for 7 min at 4000 r.p.m
7. Resuspend the cells in 200-250 l of PBS containing 2% FBS
8. Place the cell suspension to a FACS tube with 2ul of stock PI (1mg/ml) solution in the bottom. In this step and the follow to mantein on ice and dark.
9. Fluorescence emission of samples is analyzed by Flow Cell Cytometer (FACS; Becton Dickinson, Mountain View, CA) in the FL-3 channel with the Cell Quest program.
On the FACs from the Lab Inmunobioquimica (Facultad de Cs. Qcas y Farmaceuticas), the settings for HT-29 cells used in December 2001 were:
Detectors/Amps
Threshold
DetectorVoltAmp gainMode
FSH48
P1FSCE00 1.49
Line
SSC
P2SSC249 1
Log
FL1
P3FL1460 1
Log
FL2
P4FL2250 1
Log
FL3
P5FL3385 1
Log
Example of Results:
control cells, 5% serum
M1: hipodyploid population
Vicky Monardes, Jan 2002
IN SITU CASPASE-3/CASPASE-8 ASSAY BY FACS
1. A20 and Jurkat cells (0.5x106/ml) were incubated in complete medium with FasL (100 ng/ml) for 4 h at 37C in a total volume of 500 (L. For the inhibition experiments, cells were pre-incubated for 30 minutes with zVAD-fmk (5 (M).
2. Subsequently, cells were harvested by centrifugation at 3.000 r.p.m. for 7 min at room temperature (This step is necessary to eliminate all culture medium and thereby avoid dilution of the substrate).
3. Resuspend the cell pellet in 15-20 (l of each substrate and incubated for 1 h in the presence of caspase-3 substrate FAM-DEVD-fmk (Promega) or the caspase-8 substrate FAM-LETD-fmk (Intergen) at 37C and 5% CO2. Every 15 min resuspend the pellet with care.
4. Add 1 ml of PBS 1x and centrifugate the cells at 3.000 r.p.m. for 7 min. Repeat the washing step one more time.
5. Resuspend the pellet in 300 (l PBS 1x and analyze the fluorescence emission by Flow Activated Cell Sorter (FACS; Becton Dickinson, Mountain View, CA) using the Cell Quest program. The subtrates are measured with the FL-1 channel.
For more details see The complete CaspaTag manual, Intergen, caspase activity kit.
www.intergenco.com
Claudio Hetz, Jan 2002
VIABILITY ASSAYS BY FACS
Seed 100 (l of A20 cells (0,5 x 106 cell/ml) in a 96 wells plate.
Add FasL to final concentration of 100 ng/ml and incubate the cells for 16 h at 37C. Harvest the cells and wash each well with 200 (l of PBS plus 2% FCS. Centrifuge the cells for 7 min at 3.000 r.p.m. Note: For DNA content analysis:
2. Cells were harvested and the pellet were resuspended vigorously by hand and then the tubes were put on ice.
3. The cells were resuspended in methanol 95% (-20C, 1ml for each aliquot of 1 x 106 cells) adding the methanol drop by drop mixing gently with a vortex.
4. Incubate the cells for 10 min at 20C. This step allows subsequent storage of cells for one week.
Resuspend the cells in 250 (l of PBS containing 2% FCS for FACS analysis and transfer the cell suspension to a FACS tube.
Stain the dead cells with 10 (g/ml of propidium iodide (PI) to determine cell viability. Samples containing roughly 1x104 cells were analyzed by FACS in the channel FL-3 using the Cell Quest program.
Claudio Hetz, Jan 2002
ASSESSMENT OF CHROMATIN CONDENSATION AND MORPHOLOGICAL CHANGES
1. Cells were treated as described previously (viability assays), and stained after 16 h cells with PI (1 (g/ml) for 5 min in PBS on ice.
2. After washing twice in PBS 1x, cells were treated with glycerol-DABCO and viewed by an Axiovert 100 Carl Zeiss confocal microscopy upon excitation at 488nm using a 610nm emission filter. As a control, cells were permeabilized by addition of 500 (l ice-cold ethanol and incubated for 10 min at 20C before staining with PI.
Claudio Hetz, Jan 2002
QUANTIFICATION OF PHOSPHATIDYLSERINE EXPOSURE BY FACS
Presence of phosphatidylserine in the outer leaflet of the plasma membrane was detected following instructions of the manufacturer by flow cytometry using FITC-coupled annexin V (annexin V-FITC) using the ANNEXIN-V-FLUOS STAINING KIT (Roche, 1858777).
1. Seed 100 (l of A20 cells (0,5 x 106 cell/ml) in a 96-well plate.
2. Add FasL to final concentration of 100 ng/ml and incubate the cells for 16 h at 37C.
3. Harvest the cells and wash each well with 200 (l of PBS plus 2% FCS. Centrifuge the cells for 7 min at 3.000 r.p.m. (This is necessary to eliminate all culture medium and thereby avoid dilution of the probe).
4. Resuspend the cells in 25 (l of the following solution:
Propidium iodide
0.5 (l
Annexin-V-FLUOS
1 (l
Binding buffer
24 (l
As controls and to calibrate the cytometer (compensation of fluorescence), stain the negative (non-treated cells) and the positive (i.e. FasL-treated cells) controls with either Annexin or PI, respectively.
5. Incubate the cells for 30 min at room temperature in darkness. Every 10 min resuspend the pellet with care.
6. Add 250 ml of binding buffer (at 4 C) and analyze the samples by FACS. Annexin-V-fluos is measured in the FL-1 channel and PI for this experiment in the FL-2 channel.
Claudio Hetz, Jan 2002
DNA FRAGMENTATION ASSAY
Method
After the respective incubations, cells are washed twice in 400(l cold PBS following centrifugation for 5min at 3000-4000 rpm (Eppendorf centrifuge). To the pellet of washed cells add 100(l of cold PBS and 100(l of phenol:chloroform:isoamyl alcohol (25:24:1). Mix vigorously for 30sec and centrifuge 5min at 13000 rpm and 4oC (if possible). Separate phases should be visible after centrifugation. If not, repeat this step. Remove very carefully upper aqueous phase and put in a fresh Eppendorf tube. Mix 17(l of this supernatant with 2ml RNAse Buffer (Buffer 3 from Gibco-BRL) and 1(l RNAse A (1mg/ml) and incubate for 30 min at 37oC. Then add 4(l of 6x DNA loading buffer, load onto a 2%agarose gel and run at 70-100V for 15-30min or until the blue dye front migrates 3/4 down the gel. View on transilluminator with UV source.
Based on protocol by Martin Hunn
Established in Chile by Patricio Rojas, July 2001
CASPASE-3 ACTIVITY ASSAY (CHROMOGENIC)
After the respective incubations, cells are washed twice in 400(l cold PBS following centrifugation for 5min at 3000-4000 rpm (Eppendorf centrifuge). Cell pellets are left on ice until processed further. To cells add 100l of CHAPS buffer containing protease inhibitors and 1(l of the caspase-3 substrate pNA-DEVD (final conc. 100(M) and resuspend by pipetting up and down 5-6x (N.B. solution becomes viscous due to liberation of DNA). Incubate for 30min at 37oC and then centrifuge 15min at 13000rpm and RT. Remove supernatant, place in ELISA plate and continue incubation at 37oC for 1h. Read in ELISA plate reader at 405nm (best reader in Immunology or Parasitology).
Note.
1) use CHAPS buffer with inhibitors and substrate to determine background value.
2) Once cells have been washed with PBS they may be stored as a frozen pellet at 20oC.
Based on protocol by Martin Hunn
Established in Chile by Patricio Rojas, July 2001
C: BIOCHEMISTRY34TX-114 EXTRACTION
35GEL RECIPES
36DETERMINATION OF PROTEIN CONCENTRATION WITH BCA
37PROTEIN CONCENTRATION DETERMINATION WITH BIO-RAD KIT
38CAVEOLAE PURIFICATION FROM MDCK CELLS IN SODIUM CARBONATE
40CHLOROFORM:METHANOL PROTEIN PRECIPITATION FOR GEL ELECTROPHORESIS
4114C-CHOLESTEROL LABELING OF CELLS BEFORE FRACTIONATION ON SUCROSE GRADIENT
43PREPARATION OF MOUSE COLON CRYPTS
44CYTOKERATIN PURIFICATION FROM TISSUES
45IN GEL MAPK ASSAY (ERK)
47IN GEL MAPK ASSAY (JNK)
49PHOSPHOAMINO ACID ANALYSIS: MARK KAMPS'S METHOD
51POURING GELS
52AMIDOSCHWARTZ PROTEIN DETERMINATION
53PETERSON PROTEIN ASSAY
54SILVER STAIN PROCEDURE BIO-RAD KIT
55SUBCELLULAR FRACTIONATION
58EXTRACTION OF TISSUE CULTURE CELLS AND T-CELLS
59T-CELL PREPARATION
60T-CELL LABELING WITH 35S METHIONINE/CYSTEINE
61TISSUE ISOLATION
62TISSUE EXTRACTION
63PREPARATION OF BAL 1000X STOCK
6416-BAC/SDS-PAGE: A TWO-DIMENSIONAL GEL ELECTROPHORESIS
67BIOTIN (NHS) LABELLING OF PROTEINS
68BRADFORD PROTEIN ASSAY
69STOCK SOLUTIONS FOR RIPA BUFFER
70BUFFER TRITON TO WASH IMMUNOPRECIPITATES
70PBS (PHOSPHATE BUFFER SALINE)
70CM (CULTURE MEDIA)
71OTHER BUFFERS
72HEPES BUFFER
72TN BUFFER
72CHAPS EXTRACTION BUFFER
73PREPARATION OF GST-PBD BEADS (FOR CDC42 AND RAC ASSAYS)
74RAC1 AND CDC42 ACTIVITY ASSAYS
75STIMULATION OF ....... CELLS (IP AND ENDOF TREATMENT)
76CHOLESTEROL EXPERIMENT: STIMULATION OF EL-4 CELLS AFTER CHOLESTEROL DEPLETION (ANTI-PY BLOT)
78GEL SCANNING
79IODOBEAD PROTEIN IODINATION
80IODOGEN PROTEIN IODINATION
81KINASE BUFFER
82LABELLING OF ASTROCYTES MEMBRANES WITH BIOTIN (NHS)
83MEASURING ESTERASE ACTIVITY OF ACROSIN
84SPERM MEMBRANE PREPARATION.
85MEMBRANE PREPARATION
86PREPARATION OF GST-RBD (FOR RHOA ASSAYS)
87RHOA ACTIVITY ASSAYS
88SPERM MEMBRANE PREPARATION
89SURFACE LABELLING (NHS-SS-BIOTIN) OF EL-4 CELLS
90SURFACE LABELLING OF ASTROCYTES (DI-TNC1) WITH BIOTIN (NHS)
91SURFACE LABELLING OF EL-4 CELLS WITH BIOTIN (NHS)
93THY-1 PURIFICATION FROM EL-4GB RIPA EXTRACTS
94STRIPPING BLOTS
95NITRITE MEASUREMENT ASSAY
97ISOLATION OF MITOCHONDRIA
98PURIFICATION OF DIGS
101PURIFICATION OF LIGHT MEMBRANE FRACTIONS BY TRITON X-100 EXTRACTION IN SUCROSE GRADIENT
105DOT BLOTTING WITH CHOLERA TOXIN, GM1 DETECTION IN RAFT FRACTIONS.
TX-114 EXTRACTION
1. Material
TBS 10X0.1 M Tris-HCl pH 7.4
10 mM EDTA
1.4 M NaCl
12, 1 and 0.06% TX-114 in TBS (Serva, Nr. 37243)
LAPleupeptine5 mg/ml (Fluka Nr. 62070)
pepstatine5 mg/ml (Fluka Nr. 77170)
antipain5 mg/ml (Fluka Nr. 10791)
CLB0.5% Nonidet P-40 (NP-40, Fluka Nr.74385)
0.5% deoxycholic acid (Sigma Nr. D-6750)
25 mM Tris-HCl pH 8.2
50 mM NaCl
0.01% NaN32. Protocol5 x 106 cells are lysed in 1 ml of cold TX-114 + 4.4 l LAP at 4 C (107 cells in 2 ml of the same mix) inside 2ml Eppendorf tubes on ice for 1 hour. Mix the tubes from time to time.
Eliminate the nucleus by centrifuging the tubes 5 minutes at 3000 RPM in the cold room (Eppendorf centrifuge).
Rescue the supernatant and warm it for 2 minutes at 32 C.
Then centrifuge 1 minute the tubes at 6500 RPM in an Eppendorf centrifuge placed at RT.
Separate the two phases.
Resuspend the detergent phase (about 50 l) in 1 ml of cold 0.06% TX-114 and add to the aqueuse phase (about 1 ml) 50 l of cold 12% TX-114.
Warm both detergent and aqueuse phases at 32 C for 2 minutes.
Centrifuge 1 minute the tubes at 6500 RPM
Separate the two phases and pull both detergent respectively soluble fractions together in the same tube.
3. Reference
1. Bordier, C. (1981). Phase separation of integral membrane proteins in Triton-X 114 solution. J. Biol. Chem. 256, 1604-1607.GEL RECIPES
(Enough for 2 minigels)
Lower Gel:
4%6%8%10%12%12.5%15%
H2O6.165.494.824.153.483.322.50
Acryl/Bis1.332.052.683.354.024.185.00
Lower Tris2.502.502.502.502.502.502.50
10% APS0.030.030.030.030.030.030.03
TEMED0.010.010.010.010.010.010.01
Upper Gel:
H2O3.25
Acryl/Bis0.50
Upper Tris1.25
10% APS0.02
TEMED0.01
Solutions:
Acryl/Bis (30%):
29.2 g Acrylamide (Biorad 161-0101, electrophoresis purity),
0.8 g BIS (N, N' methylene-bis-acrylamide, Biorad 161-0200, electrophoresis purity)
100 ml H2O.
Make in a dark bottle, store 4 C in dark.
Lower Tris: 1.5 M Tris, pH 8.8
0.4% SDS
Upper Tris: 0.5 M Tris, pH 6.8
0.4%SDS
10% APS: Ammonium persulfate, electrophoresis grade. Either make fresh just before use or store frozen in tiny aliquots (100-150 l) and never re-use.
TEMED: Biorad 161-0800. Store 4 C, keep tightly covered and on ice when using.
RUNNING BUFFER 10x: 30g Tris (0.25 M), 174g glycine (2.32M) per litre. Final pH after dilution 8.3. For electrophoresis add 10ml 10% SDS per litre of chamber buffer
SAMPLE BUFFER 2x: 10ml glycerol 87%, 5ml beta-mercaptoethanol, 30ml 10% SDS, 12.5ml Upper-Tris per 100ml final volume. Add spatula tip of Bromphenolblue and shake well to dissolve.
DETERMINATION OF PROTEIN CONCENTRATION WITH BCA
Features
- linear working range for BSA from 20-2000 g/ml
- compatible with most ionic and nonionic detergents
- interfering substances100 mM EDTA
1mM DTT
20 % ammonium sulfate
Material and Solutions
BCA Protein Assay Reagent (Pierce N 23225)
Reagent A: Sodium carbonate and bicarbonate, BCA detection reagent and sodium tartrate in 0.1 M NaOH
Reagent B: 4% CuSO4 5H2O
Albumine Standard: 2 mg/ml
Microtiter plates (Dynatech Lab. N 001-010-2801 are the cheapest)
Method
Mix 200 l of reagent B with 10 ml of reagent A. This A+B solution is stable for 24 hours.
Follow the indication of the table to obtain appropriate BSA dilutions to establish a standard curve.
Table 1: Preparation of the diluted BSA standard
Vol. of the BSA to add Vol. of diluent to add Final BSA Conc.
300 l of (Stock) 0 l2000 g/ml
375 l of (Stock)125 l1500 g/ml (A)
325 l of (Stock)325 l1000 g/ml (B)
175 l of (A)
175 l750 g/ml (C)
325 l of (B)
325 l500 g/ml (D)
325 l of (D)
325 l250 g/ml (E)
325 l of (E)
325 l125 g/ml(F)
100 l of (F)
400 l 25 l g/ml (G)
Then mix, at least in duplicate, directly in the ELISA plate, 10 l of each BSA dilution or 10 l of the sample to be analysed (make two to three different dilutions) with 200 l of solution A+B.
Wrap the plate with parafilm and incubate it 30 minutes at 37C (GP's lab).
Read OD at 562 nm.
PROTEIN CONCENTRATION DETERMINATION WITH BIO-RAD KIT
To prepare
Standard curve
In Eppendorff tubes pipette the following volumes in l:
BSA 1.4 mg/ml0246810152030
H2O400398396394392390385380370
Final concentrations:
g/ml proteins0123457.51015
Samples
Prepare different dilutions of the samples to be analysed in Eppendorf tubes to a final volume of 400 l.
Reaction
Add to all samples 100 l of 5x of Bio-Rad reagent, vortex and leave for 15 minutes at RT.
Transfert 200 l of all reactions in an ELISA plate and read OD at 620 nm.
CAVEOLAE PURIFICATION FROM MDCK CELLS IN SODIUM CARBONATE
Material and solutions
confluent MDCK cells in 2 plates of 150 mm (50-100.106 cells)
glass loose-fitting Dounce homogenizer B (cell culture shelf, annoted MR )
Polytron tissue grinder (JP Kraehenbul's lab)
Sonicator (5 floor)
Beckmann centrifuge tubes 14x95 mm
4 ml poly-propylen tubes (Milan)
rubber policeman
PBS
500 mM Sodium Carbonate pH 11
5.3 g Na2CO3
H2O to 100 ml
(adjust pH with HCl 10 N)
MBS pH 6.525 mM Mes (Sigma)6.25 ml1 M
150 mM NaCl
7.5 ml5 M
H2O to 250 ml (adjust pH with NaOH 10N)
BAL 1000xBenzamidine
20 mg/ml
Antipain
4 mg/ml
Leupetin
0.25 mg/ml
90% sucrose in MBS
35% and 5% sucrose in MBS containing 250mM Na2CO3
solubilise the sucrose solution in 50 ml Falcon tubes by boilingsolutions either in a water-bath or simply heating in a micro-wave oven
MethodCell lysis:
All steps are done at 4C on ice.
Wash cells twice with cold PBS. Eliminate carefully all PBS after the last wash.
Add 1ml of cold Na2Co3 + 2l of BAL per plate and detach the cells using the rubber policeman. Pool cells from the two plates into a 15 ml Falcon tube. Homogenisation is carried sequencially in the following order using:
1) a loose-fitting Dounce homogenizer. Homogenize extremely carefully with 10 strokes. Be careful:
1 to avoid making foam by never pulling the piston out of the
liquid phase
2 to move the piston very slowly (10 sec per stroke)
2) a polytron tissue grinder: three 10 sec burst, 2.5 V
3) a sonicator: three 20 sec bursts, 2.5 Watt
Gradient preparation:
Using graduated pipetes, place 2 ml of 90% sucrose at the bottom of a Beckman tube, 4 ml of 35% and 5% sucrose in 2 Milan tubes. Note that these solutions are extremely viscous so pipeting requires a lot of precision. Keep these solutions at room temperature.
Mix exactly 2 ml of the lysate with the 2 ml of 90% sucrose in the centrifugation tube. Use a blue tip with a severed end to enlarge the aperture through which liquid are displaced upon pipetting. Pipet up and down delicately the mix until cell lysate and 90% sucrose are well mixed. Then put the tube on ice to cool it to 4C.
A discontinous gradient is formed above the homogenate by overlaying delicately first 4 ml of the 35% and then 5% sucrose solution. For that, use a Pasteur pipet and add, drop after drop, the sucrose solution on the surface of the tube.
Introduce the centrifugation tubes into the SW40-Ti bucklet which is then fixed on the rotor. Insert all the bucklets on the rotor, balance them and start the centrifugation using the foolowing parameters:
- brake off
- rotor code 3
- speed 30'000 rpm
- 4C
- time 20 hrs
Fraction collection:
After centrifugation, insoluble membrane fractions are flotating round the 35 and 5% interphase. A large amount of material is also visble at higher sucrose density. An small insoluble pellet is attached to the bottom of the tube.
Collect 1 ml fractions starting from the top of the tube with a 1 ml seringe. Try also to collecte the pelet, by vortexing it in 1 ml of Na2CO3. In general, analysis of 10 l from each fraction collected is sufficient to detect the enrichment of caveolin-1 in fraction 4 and 5 by SDS-PAGE and Western Blotting.
Reference1. Song et al. J. biol. Chem. 1996, 271: 9690-9697
CHLOROFORM:METHANOL PROTEIN PRECIPITATION FOR GEL ELECTROPHORESIS
Nov. 1991
Wessel and Fluegge (1984) Anal. Biochem. 138:141-143.
For up to 400 l sample in 1.5 ml polypropylene tube:
Add 400 l methanol
100 l chloroform Mix
q.s. water (to total aqueous vol of 400 l) Mix
Spin 5 min
There will be two phases, with chloroform on the bottom and the protein at the interface. Remove the supernatant to near the interface but do not remove any of the interface.
Add 300 l methanol. Mix. Spin 5 min.
Carefully remove the entire sup (use a pasteur pipet with a drawn tip).
Place the tubes under vacuum for 10 min to remove the last traces of solvent.
Resuspend the pellet in equal volumes water and 2x sample buffer for gel electrophoresis.
14C-CHOLESTEROL LABELING OF CELLS BEFORE FRACTIONATION ON SUCROSE GRADIENT
Material and solutions
adherent or non-adherent cells (MDCK and EL-4 as representative exemples)
[4-14C]-cholesterolDu Pont NEC-0180.040Ci/l
in ethanol
Pyrex glass tube 8 ml with corresponding lid
Hamilton syringea special one is stocked in Andrew's drawer
100% ethanol
Complete cell culture medium (containing serum)
MethodLabeling medium 6-12 hours preincubation
Under sterile hood.
Rinse Pyrex tube, lid and Hamilton syringe with ethanol before pipetting the cholesterol solution (10l or 0.4Ci/set of cells) into the Pyrex tube. Rinse the Hamilton syringe once with 30-40l of cold ethanol and pool this wash ethanol together with the cholesterol into the Pyrex tube. Then rinse several times the syringe in a 5ml plastic tube countaining ethanol, which is eliminated afterwards with liquide radioactive waste.
Out of the hood but work as sterile as possible.
Dry the cholesterol solution under nitrogen (Valituti's lab) by introducing directly inside the Pyrex tube a Pasteur pipette connected to the nitrogen source. It might take 10-15 minutes. Sterilize the Pasteur pipette with ethanol before use.
Under the hood again.
Fill completely the Pyrex tube with the appropriate serum (8-9ml) containing medium (minimize air volume that could oxydize medium and thus cholesterol). Close the tube with the lid and incubate the labeling solution 6-12 hours at 37 C in the incubator.Cell labeling: 36-48 hours
First, estimate the number of cells to start the experiment knowing that after the back-extraction procedure the total cell number should represent around 10mg of total proteins (2 confluent 15 cm dishes of MDCK cells or 100-130 x 106 EL-4 cells.
MDCK: 20-25% confluent cells in 2x15cm dishes (split a confluent 10 cm dish the evening before the experiment). Wash once with PBS and add the labeling mix to the cells together with 20 ml of cold medium per plate (Final concentration: 8nCi/ml)
EL-4: 15 x 106 cells. Resuspend cell pellet with 40 ml of medium + the labeling mix (Final concentration: 8nCi/ml)
Leave the cells to grow in incubator for 48 hours.
Back-extraction: 6-12 hours
MDCK: Suck medium into radioactive waste and wash cells once with PBS. Add fresh cold medium for a 6-12 hours incubation.
EL4: After pelleting the cells continue as for MDCK.
Next day, wash cells and proceed for DIG's isolation
Schedule example
Friday morningPreparation of labeling solution
Incubation up to the evening (10-12 hours)
Friday eveningAddition of labeling solution to the cells
Cell labeling 48 hours
Sunday eveningWashing of cells
Back-extraction (12 hours)
Monday morningDIG's isolation or other experiment
Radioprotection
The radioactivity used is very low but some precautions should be taken.
1. Protect the working areas.
2. Labeling solutions together with the ethanol used to wash the Hamilton syringe are collected in a bottle for radioactive waste.
3. All plastic and biological material which may contain radioactive traces that can be disposed in the normal waste but should first first, sealed in a plastic bag.
CommentDepending on the method of preparation to isolate caveolae-like domains, the cholesterol distribution after external labelling may vary. Using the sodium carbonate method most (80%) [14C]-cholesterol is recovered in the light fraction, while with the detergent method, recovery in this fraction is 40%.
PREPARATION OF MOUSE COLON CRYPTS
1Take colon from mouse and place immediately in PBS.
2Cut open the colon longitudinally and rinse out faeces with PBS.
3If intending to culture the crypt cells, place colon(s) in a solution of 0.04% sodium hypochlorite (= ~1% commercial bleach) in PBS for 20 mins at R.T. If not culturing, this step can be omitted.
4Place colon(s) in 10 ml sterile digestion mix (sterile if intend to culture cells afterwards) comprising 3 mM EDTA (111.6 mg) and 0.5 mM DTT (7.7 mg) in 100 ml PBS, for 60-90 mins at R.T or 4oC.
5Remove colon(s) from digestion mix and rinse gently once or twice with a total of 20 ml PBS.
6Place 10 ml PBS in 30 ml tube with colon(s). Cap the tube and shake lightly for a couple of seconds. Draw off PBS and transfer to 10 ml tube marked "1".
7Repeat step"6" but this time shake the tube VIGOROUSLY for about 5 seconds. Transfer to tube marked "2". This will contain an almost pure preparation of intact epithelial crypts - take a drop and check in the microscope
8Step "7" can be repeated to release more epithelial cells, but the crypts may be broken up at this stage (which doesnt matter for an RNA prep. so long as the there is not significant stromal contamination).
9Spin tubes 1, 2 & 3 at 400 rpm with brake, for 5 min at 4oC.
10Tip off PBS and resuspend crypts as appropriate for your needs.
This procedure can also be followed to release crypts from human biopsy material. (Human crypts are longer than those from the mouse).
CYTOKERATIN PURIFICATION FROM TISSUES
(Modification of protocol from E. Reichmann/W. Franke for culture cells)Make buffers the day before and store at 4C. The amounts of components indicated is for a buffer volume of 500ml.
Low Salt Buffer
High Salt Buffer
Tris
0.6g
0.6g
NaCl
9.08g
9.08g
Triton-X100
5g
5g
EDTA
0.93g
0.93g
KCl
55.9g
pH to 7.6 with NaOH (10M) - otherwise EDTA does not dissolve.
Cut tissue into small pieces with a fine pair of scissors. Add 10-fold excess over tissue weight of low-salt buffer, together with PMSF and Benzamidine.
Sonicate 3x10'' with probe sonicator at power level 2-3.
Dounce homogenize 10x - sonicate again 15" at power level 2-3.
Spin in clinical centrifuge at 5000 rpm, 4C for 5 min.
Homogenize again briefly with the Dounce homogenizer 5x in the presence of high-salt buffer. Volume might have to be doubled here, since in some cases solution will now become very viscous.
Spin at 500 rpm, 5' to remove collagenous material. Particularly in the case of tissues like lung, this might be necessary.
Extract on a rotating shaker for 30 min. or more at 4C.
Collect cytokeratins by centrifugation for 30 min. at 5000 rpm, 4C in clinical centrifuge. Carefully remove supernatant, preferably with a pasteur pipette to avoid moving weakly attached pellet.
Resuspend cytokeratin pellet from about 1-1.5g of tissue in 1 ml of PBS and split up between 10 eppendorf tubes. Spin again 5 min. at 13000 rpm, remove supernatant fluid and freeze pellets at -20C.
Each pellet, when completely solubilized in sample buffer should yield enough protein for 2-4 lanes with roughly 10-20 ug of cytokeratin per lane. Two major bands of 53 to 55 kDa are visible above the actin contamination present around 50 kDa.
(N.B. cytokeratin pellets from lung are not easily solubilized even in sample buffer, while the corresponding proteins from brain are).
IN GEL MAPK ASSAY (ERK)
Protocol from Felipe Sierra (developed for Splenocytes)
1. Cell Stimulation
- wash cells in 1x serum-free RPMI medium
- resuspend cells at 5x106 cells/ml in serum-free medium and aliquot 3ml into each plate
- equilibrate at 37C, 5% CO2 for 1-2 hours
- add 5% fetal bovine serum and stimulate, e.g. with PMA (50 ng/ml) plus A23187 (1ug/ml)
2. Whole Cell Extract Preparations
- resuspend cells in 100ul lysis buffer (15x106 cells/sample)
- shake the cell suspension at 4C for 30 min
- clarify by centrifugation at 12000xg for 10 min
- collect the supernatants (WCE)
- measure protein by the Bradford method (BioRad)
3. Preparation of gel containing myelin basic protein (MBP)
Separating gel (12.5%)
ddH2O
1.5ml
1.5M Tris pH 8.8/ 4mM EDTA
1ml
MBP (10mg/ml in pre-kinase buffer
200ul
(final conc. 0.5 ug/ml)
10% SDS
40ul
40% acrylamide/bis (29:1)
1.25ml
10% APS
20ul
TEMED
4ul
Stacking gel
the same as for a normal SDS-PAGE gel
4. Electrophoresis
- load reduced samples as usual (10ug/sample) along with pre-stained markers
- run at 200V for about 60-80 min (until prestained markers indicate that your kinase of interest is in the middle of the gel. N.B. proteins run slower than in normal gels
5. Removal of SDS
- wash gel 3x in 100ml propanol buffer at room temperature (20 min each)
- equilibrate for 1hr in 100ml ERK buffer with continuous agitation
6. Denaturation
- incubate gel for 1hr at RT with continuous agitation in 100ml denaturing buffer
7. Renaturation
- incubate the gel in 100ml renaturing buffer with gentle agitation overnight at 4C.
- the next day, wash at least another 3x in 100ml of the same buffer
8. Kinase Assay
- equilibrate the gel in 15ml pre-kinase buffer with gentle agitation for 1hr RT
- the kinase assay is done by incubating the gel for 1hr at RT in 15ml kinase buffer containing 20mM ATP and 100uCi (32P-ATP)
9. Detection
- wash the gel in 100ml wash buffer at RT 30min-1hr each time, then leave it washing overnight
- the next day, continue washing at least 3 times in 100ml of the same buffer
- autoradiography as usual
10. Solutions
Lysis buffer
25 mM Hepes pH 7.7
0.3 M NaCl
1.5 mM MgCl2
0.2 mM EDTA
0.1% Triton X-100
before use add:
2mM NaPPi
0.5mM DTT
20 mM b-glycerophosphate
5 ug/ml leupeptin
10 ug/ml aprotinin
100 ug/ml PMSF
0.1 mM sodium orthovanadate
Propanol buffer
20% 2-propanol in 50 mM Tris-HCl pH 8.0
ERK buffer
50 mM Tris-HCl pH 8.0
5 mM DTT
Denaturing buffer
6M guanidine-HCl in ERK buffer
Renaturing buffer
ERK buffer containing 0.04% Tween 20, 2mM EDTA
Pre-kinase buffer
20 mM Hepes pH 7.6
20 mM MgCl2
2 mM DTT
5 mM b-glycerophosphate
0.1 mM Na3VO4
Wash buffer
5% (w/v) trichloroacetic acid
1% NaPPi
AQ, June 98
IN GEL MAPK ASSAY (JNK)
Protocol from Felipe Sierra (developed for Splenocytes)
1. Cell Stimulation
as for ERK assay
2. Whole Cell Extract Preparations
as for ERK assay
3. Preparation of gel containing myelin basic protein (MBP)
Separating gel (12.5%)
0.75 mm 1mm comb
ddH2O
1.65ml2.15ml
1.5M Tris pH 8.8/ 4mM EDTA
1ml
1.25ml
GST c-Jun to final conc of 60 ug/ml
x ml
x ml
10% SDS
*40ul
*50ul
40% acrylamide/bis (29:1)
1.25ml1.55ml
10% APS
20ul
25ul
TEMED
4ul
5ul
$(* since the eluate solution for GST c-Jun contains SDS, make sure the final concentration of SDS is about 0.1%. The indicated values are correct if no additional SDS is present)
Stacking gel
the same as for a normal SDS-PAGE gel
4. Electrophoresis
same as for ERK, but load 30ug/sample
5. Removal of SDS
- wash gel 2x in 100ml propanol buffer at RT with continuous agitation (20 min each time)
- equilibrate for 2x in 100ml Jnk buffer at RT with continuous agitation (20 min each time)
6. Denaturation
- incubate gel 2x in 100ml denaturing buffer with continuous agitation (20 min each time)
7. Renaturation
- discard half of last volume (50ml), add 50ml renaturing buffer, continue shaking for 15min
- repeat this dilution step 3 or 4 times
- wash the gel with 100ml renaturing buffer overnight at 4C
- the next day, wash again once in 100ml of the same buffer
8. Kinase Assay
- equilibrate gel in 15ml pre-kinase buffer with gentle agitation for 30 min at 4C
- the kinase assay is done by incubating the gel for 2hr at 30C in 15ml kinase buffer containing 20mM ATP and 100uCi (32P-ATP)
9. Detection
- same as for ERK
10. Solutions
Lysis buffer
same as for ERK
Propanol buffer
20% 2-propanol in 50 mM Hepes pH 7.6
JNK buffer
50 mM Hepes pH 7.6
5 mM b-mercaptoethanol
Denaturing buffer
6M urea in JNK buffer
Renaturing buffer
JNK buffer containing 0.05% Tween 20
Pre-kinase buffer
same as for ERK
Wash buffer
same as for ERK
AQ, June 98
PHOSPHOAMINO ACID ANALYSIS: MARK KAMPS'S METHOD
...Recipes for the buffers are at the end of this protocol.
1. Label your protein with 32Pi. Then subject the protein to SDS polyacrylamide gel electrophoresis and transfer your gel-fractionated protein to Immobilon-P.
Neither nitrocellulose nor nylon will work!
Keep the membrane wet and wrap in Saran wrap.
Add radioactive markings for orientation of film later
Expose to film.
2. Cut out band of interest, re-wet in methanol, rinse well in water and place in a screw-cap tube containing 150 ul 5.7 N HCl or enough to submerge your piece of membrane.
3. Incubate in 110C oven for 60 min.
4. Microfuge sample full speed for at least 1 min.
5. Transfer supernatant to new tube and lyophilize on Speedivac. (It takes about 3 hr.)
6. Resuspend in H2O, and microfuge 5 min. Spotting more than 3 ul is tedious, so don't use much H20. On the other hand, I wouldn't use much less than 6 ul so as to have good recovery.
7. Spot 1 ul PAA standards (about 0.3 ug each of PSer, PThr, and PTyr) on a cellulose thin layer chromatography plate and then spot your sample. We use 0.1 mm EM cellulose plates
You can spot the whole sample if you are skillful and have no choice because you don't have many counts.
Spot 0.25-0.30 ul at a time and dry with house air, blown through a plugged pipet, between applications.
How do I wet the plate? We use a blotter that has four, 2 cm, circular holes cut out of it, one for each origin. We cut the holes with a sharp cork borer. The blotter can be made from a good grade of blotting paper, or two layers of Whatmann 3MM sewn together. It should be wet, but not dripping. For the first dimension, you wet the plate with pH 1.9 buffer.
8. Electrophorese at pH 1.9, 1.5 KV, 20 min in the first dimension.
9. Let plate air dry well.
How do I wet the plate for the second dimension? Use three rectangular pieces of Whatmann 3MM that have been wet with pH 3.5 buffer. Wet the bottom of the plate below the lower two origins. Keep the paper at least 1 cm away from where the PAAs are. Then wet the area between the 4 origins. Finally wet the top of the plate. The blotters should be quite damp, but not soaking wet.
10. Rotate 90 counter clockwise. Electrophorese at pH 3.5, 1.6 KV, 13 min in the second dimension.
The above electrophoresis times are appropriate when you are using a Salk/TVL/MBVL type electrophoresis rig. A knock off of this is sold by CBS scientific. If you are using another rig, you will have to optimize your electrophoresis times.
11. Dry plate in oven.
12. Spray plate with ninhydrin sol'n for several seconds. Incubate in oven until can see purple spots of PAA standards. 15 min should be plenty.
13. Mark the plate with radioactive ink so that you can extrapolate where the origins were and can align the film unambigously with the plate and the PAA markers.
14. Expose the plate to flashed film with a screen a -70C. Film is much preferable to the phosphorimager because it is transparent and is exactly the same size as the plate and this facilitates alignment of the film and the plate. In the rare case that you need quantification, you can use the phosphorimager.
15. After developing the film, trace the locations of the PAA standards and the radioactive ink marks onto a Xerox transpancy and save this in your notebook.
Recipes
pH 1.9 buffer.
88% Formic acid 50 ml
Acetic acid 156 ml
H2O 1794 ml
Don't use the 98% formic acid and don't adjust the pH.
pH 3.5 buffer
Pyridine 10 ml
Acetic acid 100 ml
H2O 1890 ml
(0.5 mmolar EDTA)
Don't bother to adjust the pH.
There is occasionally a problem with badly smeared PAAs during electrophoresis at pH 3.5. Something seems to leach out of the wicks and make the PAAs relatively insoluble. Tony thinks it is aluminum. Bart thinks that it's calcium. In any case, this problem can be prevented by including 0.5 mM EDTA in the 3.5 buffer.
AQ, July 1998
(from kinase database)
POURING GELS
September 1993
Clean glass plates, 1 large and 1 small per gel needed. Dry carefully.
In front of casting stand, line up sandwich with large plate next to plexiglass support, 2 spacers, and small glass in front. Tighten sandwich.
Mix in a 15 ml orange cap tube (for a 10 % gel, see gel recipes for other percentage gel volumes):
2.5 ml lower Tris, cold
4.15 ml H20
3.35 ml Acrylamide/Bis (29:1) (keep cold and dark)
30 l 10% ammonium persulfate (make small aliquots and store frozen, never reuse)
10 l TEMED (stored in refrigerator)
Mix gently and do not allow bubbles (oxygen inhibits the polymerization reaction; if there is a problem with the reaction going it is possible to add riboflavin, but it is not usually necessary for minigels. If used make into 100 l aliquots and store frozen.)
Overlay the gel with water-saturated butanol to keep the interface flat and to prevent air from inhibiting the reaction.
Remove the overlay by tipping the casting stand and removing the butanol with a pasteur pipet.
To make the upper gel, mix in another 15 ml conical tube:
1.5 ml upper Tris, cold
3.25 ml H20
0.5 ml Acrylamide/Bis (29:1)
20 l 10% ammonium persulfate
10 l TEMED
Mix as above, fill to the top of the sandwich. Gently insert clean comb until it reaches the top of the 2 spacers, check and then insert until the teeth are completely sealed.
AMIDOSCHWARTZ PROTEIN DETERMINATION
May 1, 1991
Procedure:
1. Put protein sample in a total volume of 270 l in 1.5 ml eppendorf tube.
2. Add 30 l sample solution.
3. Add 60 l 60% TCA. Vortex. Incubate at RT at least 2 min.
4. Set up filter apparatus, using a vacuum flask and millipore 60 mm 0.45 m type HA filter (mark one side with a soft pencil for orientation of spots later!) on a fritted glass filter support. With the vacuum on, dot the protein solution onto the filter using a P-200 pipetman to apply it in a small spot. The liquid will spread to a much wider area than the spot will if you are not careful. You can get 9 spots per filter.
5. Rinse the entire filter with about 4 ml of 6% TCA.
6. Immerse the entire filter in amidoschwartz staining solution. Try to keep the staining time constant between filters, 1 min is good.
7. Rinse for 30 sec in running dH20. (This step may be omitted)
8. Wash x 3 in 20 ml destain, or until the membrane background is white.
9. Dry on a paper towel, with the blue spots up.
10. Using a cork borer cut out the spots and place in 12x75 mm glass tubes.
11. Add 600 l of eluant solution.
12. Incubate10 min RT with 3x vortex. This may be speeded up by placing the tubes into a 60 C water bath for about 3 min.
13. Read at A 630 ( up to 2 hr after incubation).
The standard curve consists of 1-10 g of protein, the stock protein is 100 g/ ml BSA.
Solutions:
Sample solution:
1 M Tris-Cl, pH 7.5, 1% SDS
60% TCA
6% TCA
Amidoschwartz staining solution:
0.1% amidoschwartz 10B (napthol blue black - Sigma N3005)in methanol:acetic acid:H20 (45:10:45 by vol)
Destain:
Methanol:acetic acid:H20 (90:2:8 by vol)
Eluant solution:
25mM NaOH, 0.05 mM EDTA, in 50 vol % EtOH:
1 ml 1 N NaOH
25 l 100 mM EDTA
25 ml 100 % ethanol
23.97 ml H2O
Standards:
100 g/ml BSA in water.
PETERSON PROTEIN ASSAY
Using Pierce Micro-BCA Protein Reagent Kit
Nov. 13, 1991
Pipet protein solution into a suitable tube (glass or plastic, 12x75 or 13x100) and bring volume to 500 l with water.
Add 50 l 0.15% deoxycholate. Mix well. Allow to sit at RT for 10-30 min.
Add 50 l 72% TCA. Mix well.
Centrifuge for 30 min at 2000 rpm in the swinging bucket centrifuge.
At this time make the BCA reagent (mix in order!):
2 parts micro-BCA Reagent C
48 parts micro-BCA Reagent B
50 parts micro-BCA Reagent A
After the centrifuge run, decant the sup and remove any excess liquid.
Add 1 ml BCA reagent and mix.
Incubate at 60 C for 1 hr.
Measure absorbance at 562 nm.
Standard dilutionsStock 1 = Pierce 2 mg/ml BSA standard
l Stock 1
l Diluent
[BSA]
10
190
100 g/ml
25
175
250
50
150
500
75
125
750
100
100
1000
Standard
Volume
Diluent
Final [BSA]
100 g/ml10 l
990 l
1.0 g/ml
250
10
990
2.5
500
10
990
5.0
750
10
990
7.5
1000
10
990
10.0
750
20
980
15.0
1000
20
980
20.0SILVER STAIN PROCEDURE BIO-RAD KIT
Revised 11/91 Solutions (for 1-2 10 cm gels)
Fixative 1--40% methanol/10% acetic acid v/v200 ml
80 ml MeOH
20 ml glacial acetic acid
100 ml dH2O
Fixative 2--10% ethanol/5% acetic acid v/v
400 ml
42.1 ml 95% EtOH
20 ml glacial acetic acid
337.9 ml dH2O
Oxidizer--dilute concentrate 1/10 with dH2O
100 ml
Silver Reagent--dilute concentrate 1/10 with dH2O100 ml
Developer--Dissolve 32 gm in 1 l dH2O
200-300 ml
Stop solution--5% acetic acid v/v
200 ml
Procedure:1. Fixative 1
200 ml
30 min (can be stored here)
2. Fixative 2
200 ml
15 min
3. Fixative 2
200 ml
15 min
4. Oxidizer
100 ml
5 min
5. dH2O
200 ml
5 min
6. dH2O
200 ml
5 min
7. Silver reagent100 ml
20 min
8. dH2O
200 ml
1 min
9. Developer
100 ml
< 1 min (until turns brown)
10. Developer
100 ml
5 min
11. Developer
100 ml
5 min (if needed)
12. Stop
200 ml
5 min
Notes:
1 -- Always wear gloves when handling gels.
2 -- All staining is done in glass or ceramic containers.
SUBCELLULAR FRACTIONATION
IMPORTANT: All steps are performed at 4C (centrifugation) or on ice. Buffers are ice-cold and contain protease inhibitors.
The fractionation experiment is done with Swiss 3T3 fibroblasts, which are 70% confluent. Per condition, 6 dishes of 15 cm are needed.
Buffers:
Buffer H
25 mMTris-HCl, pH 7.4
2 mM
EDTA
10%
glycerol
+ protease inhibitor cocktail
Nuclear buffer
20 mMTris-HCl, pH 7.4
3mM
MgCl2
+ protease inhibitor cocktail
Protease inhibitor cocktail(PI)
1x BAL (bezamidine, antipain, leupeptin)
4 mM
PMSF
5 mM
DTT
2 mg/mlpepstatin
1 mM
sodium pervanadate
Preparation of BAL 1000x stock:
Leupeptin stock (Boehringer, 1017101): To 5 mg add 2 ml dd water (conc. 2.5 mg/ml)
Antipain stock (Boehringer, 1004646): To 10 mg add 1 ml dd water (conc. 10 mg/ml)
Benzamidine: To 100 mg add 1 ml dd water (conc. 100mg/ml)
The leupeptin and antipain stocks can be stored frozen at -20C. Do not freeze-thaw more than about 2x.
Mix:
220l benzamidine
880 l leupeptin stock
440 l antipain stock
760 l dd water
This mix is equivalent to BAL 1000x. Aliquot the mix into 40 Eppendorf tubes with 50-55 l per tube. Store frozen at -20C. Thaw these aliquots only once.
Procedure
Remove medium without scratching the cell monolayer by suction from the side.
Wash cells 2x with 10 ml ice-cold PBS. Be careful, do not disturb cell monolayer (loss of cells)! Remove the PBS each time.
Add another 5 ml PBS and detach cells with rubber policeman. The cell suspension from six plates is transferred to a 50 ml NUNC tube.
Centrifuge the cells for 5 min at 1500 rpm (250x g). Remove supernatant.
Resuspend the cells in 5 ml buffer H, spin down the cells, and remove supernatant.
Resuspend cells in 1.5 ml buffer H, containing protease inhibitors. The volume of the added buffer should be about 4x as much as the volume of the cell pellet. Incubate for 10 min on ice.
The cell suspension is transferred to a B pestle dounce homogenizer. The cells are homogenized by 30 pestle strokes. Transfer the total cell homogenate into 1.5 ml Eppendorf tube and put 30 l (2% of total) into separate 1.5 ml Eppendorf tube for analysis via SDS-PAGE. Test for homogenization efficiency under light microscope with Trypan Blue.
The cell homogenate is spun for 5 min at 3000 rpm (500x g). This centrifugation permits separation of the relatively heavy nuclei from the rest of the cell. The supernatant contains crude cytosol and is transferred to Beckmann centrifugation tubes (approx. 4 ml). Put 30 l of the crude cytosol aside in separate Eppendorf tube for later analysis. The pellet contains the intact nuclei and is kept on ice.
The next step requires centrifugation at very high speed in an ultracentrifuge. Note that the tubes must be precisely balanced! The crude cytosol is spun for 1 hour at 30'000 rpm (100'000 x g). This centrifugation permits separation of high-molecular membrane structures from soluble proteins found in the cytoplasm. The supernatant, which contains the cytosol, is placed in a fresh Eppendorf tube. The pellet contains the total plasma membrane fraction.
The plasma membrane pellet is washed once with buffer H. Note, be careful not to displace pellet! Remove buffer.
Then the plasma membrane pellet is resuspended in 1.4 ml buffer H containing 1% Triton X-100. Incubate on ice for 1 hour.
Resuspend once more and put 30 l aside in separate tube for later analysis. Then the tubes are centrifuged for 30 min at 30'000 rpm (100'000x g); do not forget to equilibrate tubes before spin.
Remove the supernatant, which contains the detergent-soluble plasma membrane. The pellet contains the detergent-insoluble plasma membrane.
The detergent-insoluble plasma membrane fraction are resuspended in 1.4 ml 1x sample buffer containing reducing agent (DTT).
Intact nuclei are incubated in 2x PCV nuclear buffer containing 0.05% Triton X-100 for 10 min on ice.
Nuclei are transferred to a L pestle Dounce homogenizer. The nuclei are homogenized by 10 pestle strokes.
Transfer the total nuclear extract into 1.5 ml Eppendorf tube and put 2% of total into separate 1.5 ml Eppendorf tube for analysis via SDS-PAGE.
The nuclear extract is spun for 5 min at 3000 rpm (500x g). The supernatant contains cellular membranes and transferred into a Eppendorf tube for subsequent analysis.
The pellet containing nuclei is resuspended in nuclear buffer (2x pellet volume) and is centrifuged for 30 min at 5000 rpm (1900x g) on 45% sucrose.
The nuclei pellet is resuspended in buffer H containing 1% Triton X-100 for 1 hour on ice. Then, it is spun for 30 min at 30'000 rpm (100'000x g).
Transfer the supernatant containing nuclear extract to an Eppendorf tube. Nuclear membranes in the pellet are resuspended in 1.4 ml 1x sample buffer containing reducing agent (DTT).
Controls for quality:
Lactate deshydrogenase (LDH):
LDH is a cytosolic protein. It is possible to measure the activity of LDH by following the decrease of NADH.
Pyruvate+NADH + H+ ------> L-lactate + NAD+
The optic density of NADH is measured at 340 nm.
Na-K ATPase assay:
This is a plasma membrane enzyme.
Proteins expected in fractions:
Total cell homogenate:
1592 g
100%
intact nuclei:
120 g
8%
cytosol:
596 g
40%
total plasma membrane:
120 g
7.5%
detergent-soluble plasma membrane:82 g
5%
detergent-insoluble plasma membrane:56 g
3%
EXTRACTION OF TISSUE CULTURE CELLS AND T-CELLS
The adherent cells from a large plate (about 107 cells) are washed briefly with cold PBS + protease inhibitors (BAL 1x, PMSF 1x), then scraped from the plate surface with a rubber policeman and washed again 3x in ice cold PBS with protease inhibitors. Basically the same protocol is used for T-cells except that one starts with ConA blasts, that are first spun on to a Ficol cushion to separate dead cells (about 107 cells). At this point the cell pellet may be frozen away at -70C.
To extract the cells add to each pellet 1 ml cold extraction buffer containing protease inhibitors (1x PMSF, 1x BAL). Homogenise in a Dounce homogenizer with 8-10 strokes at 4C
Sonicate for 2 min. (power input level 1-2, keep sample on ice)
Add Triton X100 (Pierce Surfact-Amps #28314) to final concentration 0.1%, vortex and leave on ice for 20-30 min.
Spin in Eppendorf cenrifuge at 4C for 30 min. Separate supernatant from pellet. Resuspend pellet in an equal volume of extraction buffer containing protease inhibitor cocktail, vortex
Make aliquots of supernatant and pellet fractions and freeze away at -70C. Do an Amido Schwarz protein determination. For Overlay assays use at least 100-150 ug of total protein per lane. For immunoblots amount may vary depending upon antigen. In the case of caveolin in T-cells or human colon carcinoma cells also at least 100-150mg are required. Only thaw aliquots once for usage
COMMENTS/OBSERVATIONS
AQ 17-5-95
SOLUTIONS
Extraction buffer:50mM HEPES pH 8, 10% ethyleneglycol, 0.2% Triton X100, 1x BHT/DTPA
BHT 1000x:butylated hydroxytoluene (Sigma B-1378) 5mg/ml in ethanol, stored at 4C
DTPA 1000x:Diethylenetriamine-pentaacetic acid (D-1133) 500mM. Adjust pH to 7 with NaOH.
BAL 1000x:10mg/ml benzamidine, 2mg/ml antipain, 1mg/ml leupeptin, stored at -20C
PMSF 100x:
100mM in ethanol, stored at 4C
PBS 10x:2g KH2PO4, 4g KCl, 160g NaCl, 28.4g NaH2PO4.2H2O per liter. After dilution pH should be 7.2 to 7.4.
T-CELL PREPARATION
Isolate spleen from normal Balb C mouse (contains about 108 B and T cells)
Homogenize in a potter (= all glass homogeniser with rounded glass pestle) in DMEM medium/5% FCS, wash cells by resuspending in same medium after centrifugation (clinical centrifuge 20C,1500 rpm); resuspend cell pellet in cold RBRC buffer for 5 min. and lyse red blood cells at 4C.
Resuspend cells in 10ml DMEM/10% FCS, let cells sediment for 10 min. at 20C and spin again at 1500rpm.
Take supernatant and wash 1x in DMEM/10% FCS. Resuspend in 10ml DMEM/10% FCS and distribute between 4x25 ml flasks (2.5ml per flask). To each flask add 2.5ml DMEM/10% FCS containing 4ug/ml ConA (Pharmacia # NO17-0450-01)
Incubate for 3 days at 37C in incubator with flasks in upright position.
After 3 days spin cells (mainly T-cells) down in clinical centrifuge, 5 min., 20C at 2000 rpm. Wash 1x by resuspending in PBS at 20C and centrifuging again as above. Resuspend pellet in 5ml PBS. Underlay PBS with Ficol (Sigma #NR 10771) at RT. Spin 10 min., 2000 rpm, 20C without brakes. Live T-cells are at interphase, dead T cells at the bottom of the tube. Collect interphase, wash 2x with PBS to remove Ficol. Yield, about 107 cells.
SOLUTIONS
RBRC: 13mM NaHCO3, 156 mM NH4Cl, 127 mM EDTA
PBS
: 1x
AQ 4-5-95
SEQ CHAPTER \h \r 1T-CELL LABELING WITH 35S METHIONINE/CYSTEINE
In the case of T-cells, after growing in DMEM complete medium containing ConA the live cells are separated from dead cells by spining them on a Ficol cushion (See T-cell preparation). The live cells accumulate at the interphase (about 107 cells after starting from a single spleen)
Collect interphase and wash 3x with methionine/cysteine-free medium (Gibco-BRL); spin each time at 2000rpm, 20C, clinical centrifuge
Preincubate in the methionine/cysteine-free medium containing 5% dialysed FCS (fetal calf serum) for 1 hour at 37C
Spin in clinical centrifuge at 2000 rpm, 20C for 5 min., take cell pellet up in 2-3ml methionine/ cysteine-free medium containing 5% dialysed FCS, to which 400uCi of 35S Meth/Cys (NEN or Amersham) are added. Incubate 4 hours at 37C in B-lab incubator
Spin cells down in clinical centrifuge as above and wash 1x with normal medium containing methionine/cysteine. To do a subsequent immunoprecipitation, lyse cells in 1ml of Crumptons Lysis buffer containing 10mM Iodoacetamide and 50 mM PMSF
SOLUTIONS
Crumptons Lysis Buffer:0.5% NP-40, 0.5% Deoxycholate, 50mM NaCl, 25mM Tris-HCl pH= 8.2
Dialysed FCS:Normal FCS, dialysed 48hrs against PBS 1x, with 2 changes of PBS
COMMENTS/OBSERVATIONS
AQ 11-5-95
TISSUE ISOLATION
Rats (n= .......) were sacrificed by............................................................... and stored at 4 C until organs were removed, which occured within 4 hours of death. The following organs were obtained: brain, lung, heart, liver, testes, ovary, kidney. Spleen were not available (removed by Tschopps group). The organs liver, kidney, heart and lung were cut into slices to fit into the screw-cap tubes. This was done on ice. Individual brains or testes, or 4 ovaries fit into a tube. In the case of brain also large falcon tubes containing 5 brains (L) were put aside for PKC preparations. All samples were immediately flash frozen in liquid nitrogen once in the tubes and subsequently stored long-term at - 70 C. Samples are only thawed once for extraction!
Date of Tissue Preparation:
Tissue
Nr. of Tubes
Usage
Brain
Lung
Heart
Liver
Kidneys
Testes
Ovaries
Comments:
TISSUE EXTRACTION
To make extracts, thaw individual tubes only once.
