Protein-protein interactions and western blotting

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Protein-protein interactions and western blotting

• MCB 130L
• Lecture 3

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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

EM of resting and activated B cells Activated
Extensive rough ER for antibody
production/secretion
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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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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

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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 antigen) 4.
Purify antigen and its binding partners by
centrifugation
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Immunofluorescence Microscopy
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ELISA(Enzyme-Linked Immunosorbent Assay)
Detection of proteins (i.e. cytokines, HIV
antigens) in samples
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This Weeks LabProtein-protein interactions in
synaptic vesicle fusion
Release of acetylcholine at presynaptic plasma
membrane
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Disruption of synaptic vesicle release by Tetanus
toxin

• Clostridum tetani
• Anaerobic soil bacterium
• Responsible for 350,000 cases/year of tetanus
  (spastic paralysis) worldwide
• Tetanus toxin blocks release of neurotransmitters
  from the presynaptic membranes Cleaves VAMP2

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The role of SNAREs in vesicular fusion events
SNARE Soluble NSF-Attachment Receptor protein
How is specificity achieved? How do membrane
fuse? SNARES v-SNARE vesicle SNARE t-SNARE
target SNARE Binding of v- and t-SNAREs mediates
docking and fusion Distinct cognate v- and
t-SNAREs mediate specificity
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The role of SNAREs in vesicular fusion events
(VAMP)
Structure of the SNARE complex Sb VAMP
(synaptobrevin) Sx syntaxin Sn1, Sn2 SNAP25.
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Stalk Hypothesis
Jahn and Scheller Nature Reviews Molecular Cell
Biology 7, 631643 (2006) doi10.1038/nrm2002
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SNAREs in intracellular membrane-trafficking
pathways
Jahn and Scheller Nature Reviews Molecular Cell
Biology 7, 631643 (2006) doi10.1038/nrm2002
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SNARE Domains
Chen and Scheller Nature Reviews Molecular Cell
Biology 2, 98-106 (2001)
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SNAREs form a tight complex
Isolated by size exclusion chromatography
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Identification of protein-protein interactions by
GST-pulldown assays
Purpose to determine which protein domains are
necessary for SNARE interactions
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Hey, whered all the mice go?
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Jahn and Scheller Nature Reviews Molecular Cell
Biology 7, 631643 (2006) doi10.1038/nrm2002
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SNAREs
/VAMP
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Size-exclusion chromatography and SDS-PAGE,
Biochemical Journal (2005) Volume 388, 75-79
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Botulinum toxin
JAMA. 20012851059-1070