Monoclonal Antibodies Milstein and Kohler, Nobel Prize 1984.

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Presentation transcript:

Monoclonal Antibodies Milstein and Kohler, Nobel Prize 1984

Antibodies can be coupled to fluorochromes

Immunoprecipitation Immunoblot (western blot) Reverse genetics ( got clone, can make ab and study genetics) Got antibody, can clone gene (Brian Seed) Immunofluorescence Microscopy Fluorescence Activated Cell Sorter (Herzenberg, 70s)

FACS Analysis

CD4 and CD8 can be used to subset cells of different developmental stages CD8 83% 1.6% 4.7% CD8 CD4 CD8

CD; clusters of differentiation CD1-CD247; cell surface staining Intra-cellular protein staining Solubilize cells before staining Intra-cellular cytokine staining Plug up golgi with brefeldin A and let secreted proteins accumilate Intra-cellular staining for phospho -specific forms of signal transducers

Purification of lymphocyte subpopulations (from most to least expensive and most efficient) Sort by FACs Purify using antibody coated beads Purify by antibody and complement Nylon wool for T cells Adherence to plastic for Macrophages

Transgenic mice

The deletion of specific genes can be accomplished by homologous recombination.

Gene knockout in embryonic stem cells enables mutant mice to be produced.

Cre lox technology allows conditional knockouts

The role of recessive lethal genes in lymphocyte function can be studied using RAG-deficient chimeric mice