Immunology Question

[Bodom]

Outline an experiment that would show that immunoglobulins contain heterodimers? I have this question in my class and I need some help on it. Any examples in detail would be helpful. Thanks a lot.

[Psycho]

If you isolated each fragment from the serum you could use the MR or electrophoresis, but this wouldn't conclusively show it.

[GDG]

How about a denaturing SDS-PAGE? Should show that your purified Ab turns into two populations of proteins having different weights. However, this does not prove that they aren't heterotetramers, or heterohexamers, etc.

[jimmydasaint]

I assume they would also use a Western blot with a specific anti-immunoglobulin produced as monoclonal antibodies (so they would be more specific). There is a method used here which could be easily copied:

 

Characterization of Proteins Produced by Virally Infected

Cells. Total cellular lysates and supernatants were subjected

to SDS/PAGE according to Laemmli (23). For studies involving

the intrinsic [35S]methionine labeling of cellular proteins,

1 ,uCi (1 Ci = 37 GBq) of [35S]methionine was added to

previously methionine-starved cells and incubated for 4 hr.

Acrylamide gels were dried, and autoradiograms were produced,

typically in 30-min exposures using Kodak XAR film

and Kronex (DuPont) intensifying screens. Western blot

analysis was carried out essentially as described (24), with an

1251I-labeled affinity-purified goat anti-mouse IgG antiserum

 

http://www.pnas.org/content/87/10/3942.full.pdf