Transcript Lecture_3. ppt - Department of Molecular & Cell Biology

Protein-protein interactions
and western blotting
MCB 130L
Lecture 3
Antibodies in the Immune System
Structure:
2 heavy chains + 2 light chains
Disulfide bonds
2 antigen binding sites
Isotypes: IgG, IgM, IgA, IgE, IgD
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
Antibody Production
1. 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
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
purify antibodies w/ affinity chromatography
using antigen attached to beads
Conformational
epitope
“epitope” = unique part of
antigen recognized by antibody
Producing antibodies to a specific antigen
Monoclonal antibodies:
• Derived from B-cell clone “Hybridoma”
• Recognize single epitope on antigen
Uses of antibodies in molecular biology
Applications:
Western blotting (Immunoblotting)
- Identification of protein antigen following SDS-PAGE
Immunoprecipitation
- Isolation of specific proteins + binding partners
Immunofluorescence microscopy
- Localization of specific proteins in cells
ELISA (Enzyme-Linked Immunosorbent Assay)
- Detection of proteins in a sample
Detection of specific proteins:
SDS-PAGE and Western blot
1.
2.
3.
4.
5.
6.
Separate proteins by SDS PAGE
Transfer proteins to membranes (i.e. Nitrocellulose)
Block non-specific sites on membrane
Incubate with primary antibody, wash
Incubate with secondary antibody, wash
Detect secondary antibody
Western blotting
From Lodish et al.
Molecular Cell
Biology 4th edition.
Indirect immunodetection
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
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
bead
protein A
primary
antibody
Immunofluorescence Microscopy
ELISA
(Enzyme-Linked Immunosorbent Assay)
Detection of proteins (i.e. cytokines, HIV antigens) in samples
This Week’s Lab:
Protein-protein interactions in synaptic vesicle fusion
Release of acetylcholine
at presynaptic plasma
membrane
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
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 tSNAREs mediate specificity
The role of SNAREs in vesicular fusion events
(VAMP)
Structure of the SNARE complex:
Sb = VAMP (synaptobrevin)
Sx = syntaxin
Sn1, Sn2 = SNAP25.
Stalk Hypothesis
Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002
SNAREs in intracellular membranetrafficking pathways
Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002
SNARE Domains
Chen and Scheller Nature Reviews Molecular Cell Biology 2, 98-106 (2001)
SNAREs form a tight complex
Isolated by size exclusion chromatography
Identification of protein-protein interactions
by GST-pulldown assays
protein of interest
GST
add binding
partner
bead
glutathione
bead
wash
elute with glutathione
Purpose: to determine which protein domains are
necessary for SNARE interactions
SDS-PAGE, Western blotting
Hey, where’d all the mice go?
Botulinum toxin
JAMA. 2001;285:1059-1070