Practical molecular biology: PROTEINS

Prof. Dr. Julia Kzhyshkowska

PD Dr. Alexei Gratchev

Prof. Dr. W. Kaminski

Protein analysis in tissues

Principles of protein detection Immunohistochemistry (IHC) Immunofluorescence (IF)

Protein Problem Molecular Weight (MW); how many forms; charge and shape;

Posttranslational modifications;

Biosynthetic pathways, half-life, degradation pathways;

Intracellular localisation; trafficking pathways

Integration in protein-protein networks; interaction with DNA, RNA, lipids;

Expression profile in cells and tissues;

Biological function: one or many, regulated or constitutive, intracellular or extracellular, ubiquitous or cell-type specific;

Proteins in pathology: biomarkers and their role in the molecular mechanism of a disease;

Therapeutic protein targeting

Protein identification

Direct sequencing (for purified protein);

MW: motility in the gel (usually for denatured proteins) or gel-filtration chromatography (usually for native proteins). These methods can be used for purified protein or protein complex with limited amount of components;

Proteomics-based approaches for complex protein mixtures, for example serum samples or cell lysates

Immunological detection (for purified proteins, protein complexes and crude material like cell lysates or tissue extracts)

Enzymatic activity

Protein quantification

Photometric detection: total protein amount in the sample

ELISA: measurement of particular protein concentration

Enzymatic reaction: quantification of activity, not the protein

FACS: relative quantification of protein amount in the cell

Western blotting, IHC, IF - semi-quantitative or qualitative

Immunological detection of the protein

Methods: Immunohistochemistry (IHC), Immunofluorescence (IF), Enzyime-Linked Immunosorbent Assay (ELISA) Western Blotting (WB), Immunoprecipitation (IP), Fluorescence Activated Cell Sorting (FACS)

Principle of recognition

primary antibody binds to specific epitope (one or several) in the protein

Principle of detection

primary antibody or secondary antibody that recognise primary antibody is labelled (examples: HRP for IHC and Western blotting, fluorescent dye for IF and FACS)

Material for IHC and IFFresh or frozen Tissue sections; Cells grown on cover slips; Cells sedimented on object glass

using cytospin centrifuge

Paraffin embedded Tissue sections

Advantages Disadvantages

Limited time of storage

Retrospective analysis is not possible

Advantages Disadvantages

Antigen-retrieval has to be designed individually for most of antigens

Only limited number of labeled primary antibodies recognize retrieved antigen

Antigens are in a good shape, and most of primary antibodies can be used

Intracellular localization studies are possible even in tissue sections

Extremely long storage time,

Retrospective analysis can be done on archive material

Methanol-Acetone FixationFix in cooled methanol, 10 minutes at –20 °C. Remove excess methanol. Permeabilize with cooled acetone for 1 minute at –20 °C. OrParaformaldehyde-Triton FixationFix in 3-4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 2-10 minutes. OrParaformaldehyde-Methanol FixationFix in 3-4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with cooled methanol for 5-10 minutes at –20 °C. OrPEM-Ethanol FixationFix in PEM buffer for 10 minutes. Rinse twice, briefly, with PBS. Permeabilize with cooled ethanol for 5-10 minutes at –20 °C

Fixation of fresh and frozen materialMethod of fixation has to be selected according to

1) the experimental task.

Examples: For simple identification of the protein in the cell: acetone fixation is sufficientFor precise identification of protein localization in the intracellular compartment PFA/triton is optimal

2) ability of the antibody to recognize fixed antigen.

Most of antibodies recognize antigens only in specific conditions. Example from our lab: MS-1 antibody recognizes stabilin-1 in acetone-fixed cells, but not in PFA fixed cells

IHC and IF: overlapping termsDirect Indirect

Cheap

Fast

Advantages Disadvantages

Only limited number of labeled primary antibodies are available commercially

Advantages Disadvantages

Takes more time, sometimes is more expensive

Additional control for the background staining is absolutely necessary

Wide range of labeled secondary antibodies are available commercially

It is always possible to design combination for double and triple staining

or enzyme

or enzyme

Controls

IHC

Background signal coming from substrate

IF

Auto fluorescence

2. Antibody-dependent non-specific signals/cross-reactions for IHC and IF:

2.1 Non-specific signal coming from antibody alone

Solution: optimization of concentration of secondary antibody (not signal has to be observed when primary antibody is not applied)

2.2 Non-specific signal coming from primary antibody. Following controls for primary antibody have to be used and concentrations have to be optimized:Isotype control for monoclonal antibodyPreimmune serum for polyclonal antibody-containing serumMatching Ig for purified polyclonal antibody

Important note: by optimization working concentrations has to be calculated NOT dilution

1. Antibody-independent of non-specific signals

Experimental design for IHC

1 2 3 4

Substrate (DAB) for IHC + + + +

1st antibody _ _+ Preimmune serum for

polyclonal ab or isotype control ab for monoclonal ab

+

Antigen- specific ab

2nd antibody

_ + + +

IHC and IF on frozen tissues: human lymph node samples

Martens, Kzhyshkowska et al, J Pathology, 2006

Identification of double positive cells by IF

Martens, Kzhyshkowska et al, J Pathology, 2006

IHC on frozen tissues: mouse tumour sections

Amount of stabilin-1+ TAM is significantly decreased in TS/A-SI-CLP tumors compared to TS/A-vector tumors

Schuiping Yin, TMR Student 2011/2012Master Thesis

IF on frozen tissues of mouse tumorAnalysis of co-expression of CD206 and stabilin-1 in TS/A-vector and TS/A-SI-CLP tumor

Stabilin-1-CD206+ and stabilin-1+CD206+ TAM appear in TS/A-vector and TS/A-SI-CLP tumors. The main phenotype of TAM in TS/A-vector tumor is stabilin-1+CD206+, while the main TAM phenotype in TS/A-SI-CLP tumor is stabilin-1-CD206+

stabilin-1+CD206+

stabilin-1-CD206+stabilin-1+CD206+

stabilin-1-CD206+

stabilin-1+CD206+

Schuiping Yin, TMR Student 2011/2012Master Thesis

Additional treatments are needed for staining of paraffin-embedded tissues

1. Deparaffinisation

2. Antigen retrieval

DeparaffinizationBefore proceeding with the staining protocol, the slides must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section.

Protocol

Place the slides in a rack, and perform the following washes:Xylene: 2 x 3 minXylene 1:1 with 100% ethanol: 3 min100% ethanol: 2 x 3 min95% ethanol: 3 min70 % ethanol: 3 min50 % ethanol: 3 minRunning cold tap water to rinse

Keep the slides in the tap water until ready to perform antigen retrieval. At no time from this point onwards should the slides be allowed to dry. Drying out will cause non-specific antibody binding and therefore high background staining

Antigen retrieval

The demonstration of many antigens can be significantly improved by the pre-treatment with the antigen retrieval reagents that break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites.

The techniques involved the application of heat for varying lengths of time to formalin-fixed, paraffin-embedded tissue sections in an aqueous solution (retrieval solution). This is called "Heat Induced Epitope Retrieval (HIER)". Another method uses enzyme digestion and is called "Proteolytic Induced Epitope Retrieval (PIER)". 

HIER PIER

Citrate buffer pH 6.0Tris-EDTA buffer pH 9.0EDTA buffer pH 8.0

Proteinase KTrypsinChymotrypsinPepsinPronase

IHC staining of paraffin-embedded human breast cancer

Expression of CD68 and stabilin-1 in breast cancer by the stages: stage I CD68 (A1 and A2); stage II CD68 (B1 an B2); stage III CD68 (C1 and C2); stage IV CD68 (D1 and D2); stage I stabilin-1 (E1 and E2), stage II stabilin-1(F1 and F2); stage III stabilin-1(G1 and G2); stage IV stabilin-1 (H1 and H2). Scale bars 100 μm (A1 to H1), 50 μm (A2 to H2).

Aida Avdic, TMR Student 2011/2012Master Thesis

Immunofluorescence/confocal microscopy on of paraffin-embedded human breast cancerAnalysis of CD68+/stabilin-1+ macrophages

Aida Avdic, TMR Student 2011/2012Master Thesis

Multiple IF

PL-FITC

Stabilin-1 staining

TGN-46 Merge

Most frequently double and triple IF are usedColor codeRed + green = yellowRed + blue = pinkGreen + blue = cyanGreen + red + blue = white

Kzhyshkowska et al, JI, 2008

Human macrophage

Human placenta

Stabilin-1

Ab CLEVER Ab F4 Merge

Multuple IHC

Multiple staining can also be done with enzyme conjugated antibodies developed with different chromogen substrates to produce the end products of different colors

IHC: principle of EnVision detection system from DAKO

Enzyme: Alkaline Phosphatase (AP) or Horseradish Peroxidase (HRP)

Polymer permits binding of up to 100 HRP molecules and up to 20 antibody per backbone

Horseradish peroxidaseThe enzyme horseradish peroxidase (HRP), found in horseradish, is used extensively in molecular biology applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule

In the presence of H202 (hydrogen peroxide) DAB (3,3'-Diaminobenzidine) is converted to an insoluble brown reaction product and water by the enzyme HRP

DAB + H202 ----------HRP----------> DAB ppt + H20

DAB ppt – insoluble, brown

IHC: New markers for sinusoidal cells in human lymph nodes

Martens, Kzhyshkowska et al, J Pathology, 2006

Literature

Current protocols in molecular biology www.methods.info www.dako.com

Questions?