monoclonal antibodies milstein and kohler, nobel prize 1984
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Monoclonal Antibodies
Milstein and Kohler, Nobel Prize 1984
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Antibodies can be coupled to fluorochromes
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• 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)
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FACS Analysis
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CD4 and CD8 can be used to subset cells of different developmental stages
CD8
83%
1.6%
4.7%
CD8
CD4
CD8
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CD; clusters of differentiation• CD1-CD247; cell surface staining
Intra-cellular protein stainingSolubilize cells before staining
Intra-cellular cytokine stainingPlug up golgi with brefeldin A and let secreted proteins accumilate
Intra-cellular staining for phospho-specific forms of signal transducers
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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
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Transgenic mice
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The deletion of specific genes
can be accomplished
by homologous
recombination.
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Gene knockout in embryonic
stem cells enables mutant
mice to be produced.
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Cre lox technology allows conditional knockouts
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The role of recessive lethal genes in
lymphocyte function can be studied using
RAG-deficient chimeric mice