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Chapter 3
Proteins and Polypeptides
George Plopper
Figure 03.13A: The light and heavy chains interact to form binding sites at the tips of
the short arms of the antibody.
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.
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.
Figure 03.14: Five classes of bonds
that stabilize protein structure.
Figure 03.15: Seven different types
of molecules can be covalently
attached to amino acid side chains.
Figure 03.16: Many proteins have more
than one binding site. Allosteric binding
sites help control the shape and function
of a protein.
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.
Figure 03.18: Proteasomes digest proteins in the cytosol.
Figure 03.19: Most
membrane proteins
and engulfed proteins
are digested by
lysosomes.
Figure 03.20: Extracellular proteins are digested by proteinases.
Methods in Cell & Molecular Biology
• Cell fractionation
– Detergents
– Centrifugation
– Gel electrophoresis
• Protein isolation and detection
– Immunoprecipitation
– Western blotting
Detergents - Amphiphiles
Sodium Dodecyl Sulfate (SDS, aka
Sodium Lauryl Sufate)
Triton-X-100
IONIC =
DENATURING
NONIONIC =
NOT
DENATURING
Centrifugation
http://www.coleparmer.com/TechLibraryArticle/30
Gel electrophoresis
• SDS Polyacrylamide Gel Electrophoresis (SDS
PAGE)
Western blotting
Sample Western Blot
Sources
• http://biomoleculaire.blogspot.com/
• http://employees.csbsju.edu/hjakubowski/cla
sses/ch331/Techniques/TechElectrophoresis.h
tm
• http://www.labome.com/review/gene/mouse
/Irs1-antibody-western-blot.html