1. Western blotting

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

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. Since we are trying to separate many different protein molecules of different shapes and sizes, we first want to denatured so that the proteins no longer have any secondary, tertiary or quaternary structure (i.e. we want them to retain only their primary amino acid structure). Consider two proteins that are each 500 amino acids long but one is shaped like a closed umbrella while the other one looks like an open umbrella. If you tried to run down the street with both of these molecules under your arms, which one would be more likely to slow you down, even though they weigh exactly the same? This analogy illustrates mass and the 3D structure of a molecule will determine how well it can move through an environment. We use SDS to denature all proteins to the same linear shape.

8. SDS PAGE gel following staining with Coomassie Brilliant Blue R-250 Estimation of Protein Molecular Weight Utilizing Protein Standards

9. Determining the Rm for individual proteins

10. SDS PAGE Determination of the MW of an unknown protein

11. 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

12. 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

13. 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