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Antibody & Western B lotting

Antibody & Western B lotting. 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

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Antibody & Western B lotting

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  1. Antibody & 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 EM of resting and activated B cells Activated: Extensive rough ER for antibody production/secretion

  4. Antibody Production 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. Producing antibodies to a specific antigen Polyclonal antibodies: Derived from multiple B-cell clones, recognize multiple epitopes on antigens Inject with antigen Linear epitope collect blood serum Conformational epitope purify antibodies w/ affinity chromatography using antigen attached to beads “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 Immunofluorescencemicroscopy - 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 Separate proteins by SDS PAGE Transfer proteins to membranes (i.e. Nitrocellulose) Block non-specific sites on membrane Incubate with primary antibody, wash Incubate with secondary antibody, wash Detect secondary antibody Western blotting From Lodish et al. Molecular Cell Biology 4th edition. Indirect immunodetection

  9. Detection of specific proteins: SDS-PAGE and Western blot • 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

  10. bead protein A primary antibody Immunopreciptation: Identification of protein-protein interactions 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. Antibody techniques ELISA(enzyme-linked immunosorbent assay) allows for rapid screening and quantification of the presence of an antigen in a sample Test for presence of herpes simplex virus antibodies in a blood sample

  14. Experiment Group 1. pAP-K6UbGLP-1 Group 2. pAP-UbGLP-1 Group 3. pAP-K6UbExendin-4 Group 4. pET26-Exendin-4-Ub-H6 Transformation into E. coli BL21(DE3) Expression in flask culture SDS-PAGE & Western blotting Compare the cells before and after induction

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