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

The use of antibodies in cell biology Antibodies bind to antigens (proteins, carbohydrates..) specifically and with high affinity Applications: Immunoblotting (western blotting) - Indentify protein antigen following SDS-PAGE Immunoprecipitation - Isolate specific proteins + binding partners Immunofluorescense microscopy - Localize specific proteins in cells

Antibody (immunoglobulin or Ig) structure and function Figure 1: Drawing of a typical antibody molecule. From Alberts Molecular Biology of the Cell, 4 th edition Structure: 2 heavy chains + 2 light chains Disulfide bonds 2 antigen binding sites Flexible hinge region Different types: IgG, IgM… Function: bind to foreign antigens with specificity and high affinity (ex: proteins from bacteria or viruses)

Antibodies are produced by B lymphocytes (B cells) Figure 2: Clonal selection theory. The immune system contains millions of B cell clones, each with a cell-surface receptor that binds to a certain antigen. Each antigen activates only those B cell clones that are already committed to respond to it. Activated B cells secrete antibodies that recognize the antigen. Figure 3: Electron migrograph of an inactive and active B cells. Activated B cells have an extensive rough ER involved in the production and secretion of antibodies.

Producing antibodies to an antigen of interest (ex. a specific protein) 1.Inject antigen (ex: purified protein of interest) into animal (ex: 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 by affinity chromatography using antigen attached to beads Producing antibodies to an antigen of interest (ex. a specific protein) Polyclonal antibodies: Derived from multiple B-cell lines, recognize multiple epitopes on antigens Linear epitope Conformational epitope

Producing antibodies to an antigen of interest (ex. a specific protein) Derived from a single B- cell clone, recognize a single epitope on antigen Hybridoma

Detection of specific proteins in a complex mixture by SDS-PAGE and immunoblotting (Western blotting) Figure 7: Western blotting. From Lodish et al. Molecular Cell Biology 4 th edition. Figure 8: Indirect immuno-detection. 1.Separate proteins by SDS PAGE 2.Transfer proteins to membranes (ex Nitrocellulose), electrophoresis 3.Block non specific sites on membrane 4.Incubate with primary antibody, then wash 5.Incubate with secondary antibody, then wash 6.Detect secondary antibody

Detection of specific proteins in a complex mixture by SDS-PAGE and immunoblotting (Western blotting) Figure 9: Detection of HRP labeled secondary antibody by chemiluminescence. HRP oxidizes a compound called luminol, which then decays through several light emitting states. Light from oxidized luminol is detected using film. Figure from Amersham Biosciences. secondary antibody horseradish peroxidase (HRP) luminol

Identification of protein-protein interactions by Immunoprecipitation 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

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

C. tetani blocks the release of neurotransmitters from the presynaptic membranes of inhibitory nerve synapses. Cleaves VAMP2. C. tetani is a anaerobic soil bacterium responsible for an estimated 200 cases of tetanus (spastic paralysis) every year in the U.S. and an additional 350,000 worldwide. Tetanus toxin

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 V and T-SNAREs bind each other (docking), Mediate fusion Distrinct cognate V and T-SNAREs for Different compartments - mediate specificity

The role of SNAREs in vesicular fusion events Figure 17: Structure of the SNARE complex. Sb=VAMP or synaptobrevin, Sx = syntaxin, Sn1, Sn2 = SNAP25. Figure 16: The structure of paired SNAREs..

Fusion of synaptic vesicles with the plasma membrane Figure 19: Neurotransmitter secretion. From the MCB 130L lab manual.

Fusion of synaptic vesicles with the plasma membrane Figure 20: Domain interactions between SNAP25, VAMP and syntaxin. (T-SNARE) (V-SNARE)