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 - AmphiphilesTriton-X-100Sodium Dodecyl Sulfate (SDS, aka

Sodium Lauryl Sufate)

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.htm

• http://www.labome.com/review/gene/mouse/Irs1-antibody-western-blot.html