Western blotting

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Western blotting
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Antibodies in the Immune System
Structure 2 heavy chains 2 light
chains Disulfide bonds 2 antigen binding
sites Isotypes IgG, IgM, IgA, IgE, IgD
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Antibodies are produced by B lymphocytes

• Clonal Selection
• Millions of B cell clones w/ specific
  cell-surface receptors
• Activation of B cell clones by specific target
  antigen
• Activated B cells secrete specific antibodies

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Antibody Production

• Inject antigen (i.e. purified protein)
• into animal (i.e. mouse, rabbit, chicken)
• 2. Animal produces antibodies that recognize
  antigen

Antigen injected more than once response
heightened in subsequent injections
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Producing antibodies to a specific antigen
Polyclonal antibodies Derived from multiple
B-cell clones, recognize multiple epitopes on
antigens
Inject with antigen
Linear epitope
collect blood serum
Conformational epitope
purify antibodies w/ affinity chromatography using
antigen attached to beads
epitope unique part of antigen recognized by
antibody
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Producing antibodies to a specific antigen

• Monoclonal antibodies
• Derived from B-cell clone Hybridoma
• Recognize single epitope on antigen

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Since we are trying to separate many different
protein molecules of different shapes and sizes,
we first want to denatured so that the proteins
no longer have any secondary, tertiary or
quaternary structure (i.e. we want them to retain
only their primary amino acid structure).
Consider two proteins that are each 500 amino
acids long but one is shaped like a closed
umbrella while the other one looks like an open
umbrella. If you tried to run down the street
with both of these molecules under your arms,
which one would be more likely to slow you down,
even though they weigh exactly the same? This
analogy illustrates mass and the 3D structure of
a molecule will determine how well it can move
through an environment. We use SDS to denature
all proteins to the same linear shape.
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SDS PAGE gel following staining with Coomassie
Brilliant Blue R-250
Estimation of Protein Molecular Weight Utilizing
Protein Standards
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Determining the Rm for individual proteins
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SDS PAGE Determination of the MW of an unknown
protein
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Uses of antibodies in molecular biology
Applications Western blotting (Immunoblotting)
- Identification of protein antigen following
SDS-PAGE Immunoprecipitation - Isolation of
specific proteins binding partners Immunofluore
scence microscopy - Localization of specific
proteins in cells ELISA (Enzyme-Linked
Immunosorbent Assay) - Detection of proteins in
a sample
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Detection of specific proteins SDS-PAGE and
Western blot

1. Separate proteins by SDS PAGE
2. Transfer proteins to membranes (i.e.
   Nitrocellulose)
3. Block non-specific sites on membrane
4. Incubate with primary antibody, wash
5. Incubate with secondary antibody, wash
6. Detect secondary antibody

Western blotting From Lodish et al. Molecular
Cell Biology 4th edition.
Indirect immunodetection
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Detection of specific proteins SDS-PAGE and
Western blot

• Detection of HRP labeled secondary antibody by
  chemiluminescence
• Electrochemiluminescence (ECL) reagent H2O2
  luminol
• HRP catalyzes breakdown of H2O2 to H2O and O2,
• Luminol is oxidized
• Light from oxidized luminol is detected using
  film
• Figures from Amersham Biosciences