protocol for native gel!!!

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum at hotmail.com)
Tue Oct 10 12:35:52 EST 2006


arikbar at pob.huji.ac.il wrote:

> I need a good protocol for native gel that will be followed by western blot. I'm
> trying to find a receptor which is composed of five different subunits and also
> has a transmembrane domain (for each subunit). I don't know how to calculate
> the pI of the full receptor so any suggestions may help.

In the simplest case a native gel is simply a Laemli gel with SDS and/or
DTT omitted from gel and sample buffer. Somewhat more advanced is the
"blue native gel" proposed by Schägger and v. Jagov (Anal. Biochem. 199
(1991) 223-231).

Acetylcholine receptor is a pentameric transmembrane protein.

pI can not be reliably calculated from primary structures despite many
claims to the contrary, because the pKa of acidic and basic groups
depends on the environment. The tabulated values refer to free amino
acids in water and do not apply to the interior of proteins. For example
the pKa of the Glu side chain carboxylate is supposed to be 4.3, but in
xylanase active site it is 6.7.




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