[PPT]PowerPoint Presentation - Department of Molecular & …mcb.berkeley.edu/courses/mcb130L/Originals/Lecture_3.ppt · Web viewTitle PowerPoint Presentation Author Laurent Coscoy

Protein-protein interactions and western blotting

MCB 130LLecture 3

Antibodies in the Immune System

Structure:2 heavy chains + 2 light chainsDisulfide bonds2 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 cellsActivated: 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

collect blood serum

purify antibodies w/ affinity chromatographyusing antigen attached to beads

Producing antibodies to a specific antigenPolyclonal antibodies: Derived from multiple B-cell clones,recognize multiple epitopes on antigens

Linear epitope

Conformationalepitope

Inject with antigen

“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

Western blottingFrom Lodish et al. Molecular Cell Biology 4th edition.

Indirect immunodetection

1. Separate proteins by SDS PAGE2. Transfer proteins to membranes (i.e. Nitrocellulose)3. Block non-specific sites on membrane4. Incubate with primary antibody, wash5. Incubate with secondary antibody, wash6. Detect secondary antibody

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

Detection of specific proteins:SDS-PAGE and Western blot

Immunopreciptation: Identification of protein-protein interactions

bead

protein A

primary antibody

Steps:1. Attach antibody to beads via protein A2. Lyse cells to release antigen and its binding partners3. Mix cell lysate + antibody-coated beads (antibody binds antigen)4. Purify antigen and its binding partners by centrifugation

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

How is specificity achieved?How do membrane fuse?

SNARES:v-SNARE: vesicle SNAREt-SNARE: target SNARE

Binding of v- and t-SNAREs mediates docking and fusion

Distinct cognate v- and t-SNAREs mediate specificity

“SNARE” = Soluble NSF-Attachment Receptor protein

The role of SNAREs in vesicular fusion events

Structure of the SNARE complex: Sb = VAMP (synaptobrevin)Sx = syntaxinSn1, Sn2 = SNAP25.

(VAMP)

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002

Stalk Hypothesis

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002

SNAREs in intracellular membrane-trafficking pathways

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 interactionsby GST-pulldown assays

GST

bead

glutathione

protein of interest

bead

add binding partner

wash

elute with glutathione

SDS-PAGE, Western blottingPurpose: to determine which protein domains are necessary for SNARE interactions

Hey, where’d all the mice go?

Botulinum toxin

JAMA. 2001;285:1059-1070