Transcript Western Blotting - Universitas Brawijaya

Concept of Ag-Ab immunological
technique
•
Using the characteristic of high affinity and
specificity of antibody, we can detect or
quantitative the antigen. Keep in mind that
the antibody is protein, can also be
recognized as an antigen. The major principle
to determine the antigen-antibody interaction
is to separate the bound form of antigenantibody complex from the free form of either
antigen or antibody.
Antibodies
Analytical Techniques
Utilizing Antibodies:
• flow cytometry
• gel electrophoresis
• immunoprecipitation (IP)
• immunoblotting
• microscopy
• immunofluorescence (IFA)
• electron microscopy
• ELISA
• antibodies bind proteins with
high specificity and affinity
• affinity chromatography
• analytical techniques
CNBr

Western Blotting
Western Blotting
• Protein denature (SDS)
• SDS-PAGE gel
electrophoresis
• Blotting (transfer)
• Blocking (BSA)
• Staining with Coomasie or
Ponceau S (checking)
• 1° Ab serum (probe)
• Washes
• 2 ° Ab serum
• Washes
• Color development
Why do proteins stick to the membrane? Hydrophobic & charge interactions.
Vertical Gel Electrophoresis
Prep and Run Samples
Example of Western Blot Result
Blot interpretation
1.
2.
3.
4.
5.
Lane 1, HIV+ serum
(positive control)
Lane 2, HIV- serum
(negative control)
Lane A, Patient A
Lane B, Patient B
Lane C, Patient C
Enzyme-Mediated Detection
Enzyme
Horseradish Peroxidase
(HRP)
Substrate
Abbrev.
Color
Diaminobenzidine
DAB
Brown
Enzyme
Alkaline Phosphatase (AP)
Substrate
Abbrev.
Bromochloroindolylphosphate
Nitro Blue Tetrazolium
BCIP (AP substrate) Purple
NBT (Enhance color)
Color
TUGAS:
1. Komposisi dan fungsi dari masingmasing bahan pada transfer buffer
2. Methode secara rinci western
blotting
Immunopreciptation:
Identification of protein-protein interactions
Steps:
1. Attach antibody to beads via protein A
2. Lyse cells to release antigen and its binding partners
3. Mix cell lysate + antibody-coated beads (antibody binds antige
4. Purify antigen and its binding partners by centrifugation
bead
protein A
primary
antibody
Immunoprecipitation
• affinity purification based on
isolation of Ag-Ab complexes
• analyze by gel electrophoresis
• initially based on centrifugation of
large supramolecular complexes
• [high] and equal amounts
• isolation of Ag-Ab complexes
• fixed S. aureus
• protein A-agarose
• protein G-agarose
Bacterial proteins that bind IgG (Fc):
• protein A (Staphylococcus aureus)
• protein G (Streptococcus)
• binds more species and subclasses
Typical IP Protocol
1. Solubilize antigen
• usually non-denaturing
• SDS + excess of TX100
2. Mix extract and Ab
3. Add protein G-agarose, etc
4. Extensively wash
5. Elute with sample buffer
6. SDS-PAGE
7. Detection
• protein stain
• radioactivity
agaroseG
Radiolabeling of Proteins
• carried out before IP
• metabolic (amino acids or
other precursors + cells)
• chemically (eg., iodination)
• IP and SDS-PAGE
• detect by autoradiography
or fluorography following
electrophoresis
• also provides information
about synthesis, posttranslational events, etc.
Western Blot vs
Immunoprecipitation
• Experimental Design
• eg., synthesis (IP)
• Ag concentration
• IP better for low
abundance proteins
• Ag solubility
• Western for insoluble
proteins
• Ab recognition
• conformational
dependent epitopes
• 4o structure
Basics of
Immunohistochemistry
03/2005
Immunofluorescence Microscopy
What is
Immunohistochemistry?
CELLULAR ANTIGENS
Sensory
Metabolic
Adhesion
Outline of Procedure
Fixwholemount, embed and section tissue (or treat as
”” preparation – small specimens only, such as
cultured cells)
Wash sections in physiological buffer, e.g. PBS
Incubate with protein solution (BSA or normal serum)
to reduce non-specific binding of antibody to
specimen (”blocking”- important!)
Incubate with antibody specific to antigen in question
(”primary antibody”). Include positive and negative
controls (!)
Wash in physiological buffer
Apply suitable detection system (see below)
Mount specimens and analyse microscopically
Fixing and Sectioning of Tissue
Common fixation methods are
chemical fixation (e.g.
paraformaldehyde) or cryofixation (i.e.
rapid freezing). Fixation serves to
preserves tissue structure and
properties of antigen
Tissues can be embedded in wax or
resins for sectioning, or cut in the
frozen state in a cryostat.
The method chosen for fixation and
sectioning are dependent on the
properties of the tissue, antigen and
the antibody used in the procedure
Immunohistochemical Detection
Methods
Through fluorescent substances
(fluorophores) –”Immunofluorescence
technique”
Through enzymatic conversion (often
by horseradish peroxidase, HRP) of a
soluble substrate (chromogen) into a
non-soluble and colourful reaction
product - ”Immunoenzyme technique”
Immunofluorescence
Fluorescence or confocal microscope
necessary for analysis of specimens
High structural resolution possible
Advanced image reconstruction (3D)
and signal quantification possible
Multiple labelling easy
Limited shelf life of labelled specimens
Method of choice for labelling of live
cells
Direct Immunofluorescence Method
Easy application, only few steps
Not very sensitive
Not very versatile, as primary antibodies
need to be directly labelled with
fluorophore
Fluorochrome-conjugated
Primary Antibody
Antigen expressing Cell
Indirect Immunofluorescence
Method
More steps involved
More sensitive
More versatile, as only secondary
antibodies need to be labelled and different
combinations of fluorophores are possible
in multiple labelling experiments
Fluorochrome-conjugated
Secondary Antibody (anti species X
Primary Antibody (from species X)
Antigen expressing Cell
Indirect Immunfluorescence
Using Biotinylated secondary
Antibody
Biotin (Vitamin B) binds with high affinity
to Avidin – thus good linker system
Very high sensitivity
Endogenous biotin may be present in tissue
– risk of background
Fluorochrome-conjugated
Streptavidin
Biotinylated
Secondary Antibody (anti species X
Primary Antibody (from species X)
Antigen expressing Cell
Multiple Immunofluorescence
Multiple Labelling of a Tissue Section
Live Labelling of Cultured Cells
Enzymatic detection methods
Brightfield microscope sufficient for
analysis of specimens
Suitable for tissue analysis at low
magnification
Resolution of subcellular structures
not as good as with fluorescence
methods, but can be combined with
electron microscopy
Unimited shelf life of labelled
specimens
Substrate reagents often
toxic/carcinogenic
Immunoezyme Labelling of Tissue
Section
ABC (Avidin Biotin Complex) Method
Biotin (Vitamin B) binds with high affinity
to Avidin – thus good linker system
Extremely high sensitivity
Endogenous biotin my be present in tissue
– risk of background
Substrate-chromogen
solution
Enzyme-conjugated
Avidin-Biotin Complex
Biotinylated
Secondary Antibody
(anti species X)
Primary Antibody (species X)
Antigen expressing Cell
Common Problems
Non-specific binding of primary or
secondary antibodies to tissue sample;
e.g. through ionic or hydrophobic
interactions or binding of antibodies to
free amino groups: „Background staining“
Cross-reactivity of antibodies with unrelated
antigens present in tissue sample
Good Resources
Dako On-line Immunohistochemistry Handbook
(http://www.dakousa.com/ihcbook/hbcontent.htm)
NIH Immunohistochemistry Protocols
(http://dir.niehs.nih.gov/dirlep/immuno.html)