Transcript Practical molecular biology

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;
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Posttranslational modifications;
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Biosynthetic pathways, half-life, degradation pathways;
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Intracellular localisation; trafficking pathways
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Integration in protein-protein networks; interaction with DNA, RNA, lipids;
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Expression profile in cells and tissues;
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Biological function: one or many, regulated or constitutive, intracellular or
extracellular, ubiquitous or cell-type specific;
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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
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ELISA: measurement of particular protein concentration
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Enzymatic reaction: quantification of activity, not the protein
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FACS: relative quantification of protein amount in the cell
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Western blotting, IHC, IF - semi-quantitative or qualitative
Immunological detection
of the protein
Methods:

Immunohistochemistry (IHC),
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Immunofluorescence (IF),
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Enzyime-Linked Immunosorbent Assay (ELISA)
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Western Blotting (WB),
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Immunoprecipitation (IP),
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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 IF
Fresh or frozen
 Tissue sections;
 Cells grown on cover slips;
 Cells sedimented on object glass
using cytospin centrifuge
Advantages
Antigens are in a good
shape, and most of
primary antibodies can
be used
Intracellular localization
studies are possible
even in tissue sections
Disadvantages
Limited time of storage
Retrospective analysis
is not possible
Paraffin embedded
 Tissue sections
Advantages
Extremely long storage
time,
Retrospective analysis
can be done on archive
material
Disadvantages
Antigen-retrieval has to
be designed individually
for most of antigens
Only limited number of
labeled primary
antibodies recognize
retrieved antigen
Fixation of fresh and frozen material
Method 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 sufficient
For 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
Methanol-Acetone Fixation
Fix in cooled methanol, 10 minutes at –20 °C.
Remove excess methanol.
Permeabilize with cooled acetone for 1 minute at –20 °C.
Or
Paraformaldehyde-Triton Fixation
Fix in 3-4% paraformaldehyde for 10-20 minutes.
Rinse briefly with PBS.
Permeabilize with 0.5% Triton X-100 for 2-10 minutes.
Or
Paraformaldehyde-Methanol Fixation
Fix in 3-4% paraformaldehyde for 10-20 minutes.
Rinse briefly with PBS.
Permeabilize with cooled methanol for 5-10 minutes at –20 °C.
Or
PEM-Ethanol Fixation
Fix in PEM buffer for 10 minutes.
Rinse twice, briefly, with PBS.
Permeabilize with cooled ethanol for 5-10 minutes at –20 °C
IHC and IF: overlapping terms
Direct
Indirect
or enzyme
or enzyme
Advantages
Cheap
Fast
Disadvantages
Only limited number of
labeled primary
antibodies are available
commercially
Advantages
Wide range of labeled
secondary antibodies
are available
commercially
It is always possible to
design combination for
double and triple
staining
Disadvantages
Takes more time,
sometimes is more
expensive
Additional control for the
background staining is
absolutely necessary
Controls
1. Antibody-independent of non-specific signals
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 antibody
Preimmune serum for polyclonal antibody-containing serum
Matching Ig for purified polyclonal antibody
Important note:
by optimization working concentrations has to be calculated NOT dilution
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 2011/2012
Master Thesis
IF on frozen tissues of mouse tumor
Analysis 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-1CD206+
stabilin-1+CD206+
stabilin-1-CD206+
stabilin-1-CD206+
stabilin-1+CD206+
Schuiping Yin,
TMR Student 2011/2012
stabilin-1+CD206+ Master Thesis
Additional treatments are needed for staining
of paraffin-embedded tissues
1. Deparaffinisation
2. Antigen retrieval
Deparaffinization
Before 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 min
Xylene 1:1 with 100% ethanol: 3 min
100% ethanol: 2 x 3 min
95% ethanol: 3 min
70 % ethanol: 3 min
50 % ethanol: 3 min
Running 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 pretreatment 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 6.0
Tris-EDTA buffer pH 9.0
EDTA buffer pH 8.0
Proteinase K
Trypsin
Chymotrypsin
Pepsin
Pronase
IHC staining of paraffin-embedded human breast cancer
Aida Avdic,
TMR Student
2011/2012
Master Thesis
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 stabilin1 (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).
Immunofluorescence/confocal microscopy on of paraffinembedded human breast cancer
Analysis of CD68+/stabilin-1+ macrophages
Aida Avdic,
TMR Student
2011/2012
Master Thesis
Multiple IF
Ab CLEVER
Ab F4
Merge
Human
placenta
Stabilin-1 staining
PL-FITC
Stabilin-1
TGN-46
Merge
Human
macrophage
Most frequently double and triple IF are used
Color code
Red + green = yellow
Red + blue = pink
Green + blue = cyan
Green + red + blue = white
Kzhyshkowska et al, JI, 2008
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 peroxidase
The 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?