chapter 3 proteins and polypeptides george plopper
TRANSCRIPT
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Chapter 3
Proteins and Polypeptides
George Plopper
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Figure 03.13A: The light and heavy chains interact to form binding sites at the tips of the short arms of the antibody.
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Antibody facts worth knowing• Like all proteins, antibodies can be denatured by
SDS and other harsh detergents• Antibodies are proteins secreted by immune cells
called B cells.• Antibodies can be isolated from serum (blood)
(polyclonal), or from a single B cell colony (monoclonal).
• Every different antibody has a unique Fab region.• All antibodies isolated from the same species of
animal have nearly identical Fc regions.
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Figure 03.13B: Each domain in the heavy and light chains is tinted a
different color for easier identification.
Protein Data Bank ID: 1IGY. Harris, L. J., Skaletsky, E., and McPherson, A., J. Mol. Biol. 275 (1998): 861-872.
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Figure 03.14: Five classes of bonds that stabilize protein structure.
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Figure 03.15: Seven different types of molecules can be covalently attached
to amino acid side chains.
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Figure 03.16: Many proteins have more than one binding site. Allosteric binding
sites help control the shape and function of a protein.
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Figure 03.17: Occupancy of an allosteric GTP binding site controls a G protein subunit's shape. Three different conformations of the subunit are overlaid for comparison.
Photo courtesy of Heidi E. Hamm and Will Oldham, Vanderbilt University Medical Center.
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Figure 03.18: Proteasomes digest proteins in the cytosol.
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Figure 03.19: Most membrane proteins
and engulfed proteins are digested by
lysosomes.
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Figure 03.20: Extracellular proteins are digested by proteinases.
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Methods in Cell & Molecular Biology• Cell fractionation– Detergents– Centrifugation– Gel electrophoresis
• Protein isolation and detection– Immunoprecipitation– Western blotting
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Detergents - AmphiphilesTriton-X-100Sodium Dodecyl Sulfate (SDS, aka
Sodium Lauryl Sufate)
IONIC =DENATURING
NONIONIC =NOT DENATURING
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Centrifugation
http://www.coleparmer.com/TechLibraryArticle/30
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Gel electrophoresis• SDS Polyacrylamide Gel Electrophoresis
(SDS PAGE)Western blotting
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Sample Western Blot
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Sources
• http://biomoleculaire.blogspot.com/• http://employees.csbsju.edu/hjakubowski/cla
sses/ch331/Techniques/TechElectrophoresis.htm
• http://www.labome.com/review/gene/mouse/Irs1-antibody-western-blot.html