Transcript File
11-7-11 Immunological Methods 1. Identification, purification and characterization of the estrogen receptor 2. Gradient centrifugation 3. Generation of polyclonal and monoclonal antibodies 4. Immunoprecipitation, ELISAs and Westerns Gradient Centrifugation Ultracentrifugation is capable of separating small molecular complexes The sedimentation coefficent (s) s = m(1 - ur)/f S = 10-13 s Mass, shape and density all affect the value of S The Estradiol-Receptor complex can be separated by gradient centrifugation Estradiol is radiolabeled to facilitate tracking A protein mixture containing the estradiol-receptor complex is layered on a sucrose gradient The gradient is centrifuged at high speed (~105 x g) Antibodies can be generated that react specifically with the estrogen receptor Polyclonal antibodies can be produced by immunizing a rabbit with a specific protein (antigen) eg the crude estradiol-receptor complex The rabbit immune system will respond by producing multiple “polyclonal” antibodies with differing specificities to the estrogen receptor Polyclonal antibodies recognize different antigenic determinants (epitopes) on the estrogen receptor Monoclonal antibodies with any desired specificity can be generated using hybridomas Immortal multiple myeloma cells are fused with antibody-producing spleen cells The resulting hybridoma cells are screened for those producing antibodies specific for the desired protein (estrogen receptor, in this case) The selected hybridoma cells are immortal and secrete a single specific “monoclonal” antibody that is easy purified Gradient centrifugation provides a screen for the desired receptor-specific hybridomas The estrogen receptor can be purified from crude cell extracts by immunoprecipitation using a monoclonal antibody Estrogen receptor-specific Mab is covalently linked to insoluble polyacrylamide or polysaccharide beads Antibody-bound beads are mixed with cytosol to bind the receptor, then centrifuged to separate from unbound proteins Highly pure receptor may then be eluted from the beads The Enzyme-Linked ImmunoAbsorbent assay (ELISA) can detect and quantify a protein (antigen) in complex mixtures A reporter enzyme (eg peroxidase) is linked to an antibody specific for the protein of interest The indirect ELISA is used to detect the presence of antibody – eg the HIV test Purified HIV core proteins are absorbed to wells in a microtiter dish Antibodies from the subject are placed in wells, then washed to remove unbound antibodies The Sandwich Elisa is used to detect antigen Antibody to a particular antigen is used to coat wells in a microtiter dish Sample containing suspected antigen is added to the well, allowed to bind to the antibody then washed out A second antibody to the antigen containing a linked reporter enzyme is added and processed Color development is proportional to the amount of antigen present Western blotting is used to detect protein antigens separated by polyacrylamide gel electrophoresis The SDS gel is electroblotted to transfer proteins to a membrane (cellulose or polymer sheet) The blot is probed with a reporter-linked antibody specific to the antigen of interest The blot is washed and processed to expose presence of the reporter-linked antibody