Transcript 390k ppt - UCLA.edu

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
CD4
83%
1.6%
CD8
4.7%
CD8
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