1. Protein-protein interactions and western blotting MCB 130L Lecture 3

2. Antibodies in the Immune System Structure: 2 heavy chains + 2 light chains Disulfide bonds 2 antigen binding sites Isotypes: IgG, IgM, IgA, IgE, IgD

3. 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

4. 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

5. 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

6. Producing antibodies to a specific antigen Monoclonal antibodies: Derived from B-cell clone “Hybridoma” Recognize single epitope on antigen

7. 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

8. 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

9. 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

10. 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

11. Immunofluorescence Microscopy

12. ELISA (Enzyme-Linked Immunosorbent Assay) Detection of proteins (i.e. cytokines, HIV antigens) in samples

13. This Week’s Lab: Protein-protein interactions in synaptic vesicle fusion Release of acetylcholine at presynaptic plasma membrane

14. 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

15. 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

16. The role of SNAREs in vesicular fusion events Structure of the SNARE complex: Sb = VAMP (synaptobrevin) Sx = syntaxin Sn1, Sn2 = SNAP25. (VAMP)

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

18. Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002 SNAREs in intracellular membrane- trafficking pathways

19. SNARE Domains Chen and Scheller Nature Reviews Molecular Cell Biology 2, 98-106 (2001)

20. SNAREs form a tight complex Isolated by size exclusion chromatography

21. 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

22. Hey, where’d all the mice go?

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

24. SNAREs /VAMP

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

26. Botulinum toxin JAMA. 2001;285:1059-1070