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

collect blood serum purify antibodies w/ affinity chromatography using antigen attached to beads Producing antibodies to a specific antigen Polyclonal antibodies: Derived from multiple B-cell clones, recognize multiple epitopes on antigens Linear epitope Conformational epitope 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 blotting From Lodish et al. Molecular Cell Biology 4 th edition. Indirect immunodetection 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

Detection of HRP labeled secondary antibody by chemiluminescence Electrochemiluminescence (ECL) reagent: H 2 O 2 + luminol HRP catalyzes breakdown of H 2 O 2 to H 2 O and O 2, 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 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

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 SNARE t-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 = syntaxin Sn1, Sn2 = SNAP25. (VAMP)

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

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi: /nrm2002 SNAREs in intracellular membrane- trafficking pathways

SNARE Domains Chen and Scheller Nature Reviews Molecular Cell Biology 2, (2001)

SNAREs form a tight complex Isolated by size exclusion chromatography

Identification of protein-protein interactions by GST-pulldown assays GST bead glutathione protein of interest bead add binding partner wash elute with glutathione SDS-PAGE, Western blotting Purpose: to determine which protein domains are necessary for SNARE interactions

Hey, where’d all the mice go?

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

SNAREs /VAMP

Size-exclusion chromatography and SDS-PAGE, Biochemical Journal (2005) Volume 388, 75-79

Botulinum toxin JAMA. 2001;285: