A fastidious contaminant

John Ladasky ladasky at leland.Stanford.EDU
Fri Mar 8 23:41:51 EST 1996


In article <199603081028.CAA01194 at net.bio.net>,
Giorgio Spagnol <spagnol at GALACTICA.IT> wrote:
>Dear fellow netters,
>When I precipitate a glycoprotein I'm studiyng with a monoclonal Ab 
>against the peptidic part, two contaminants of lower molecular weight 
>(It actually seems one, when I run nondenaturing gels) always 
>co-precipitate. Consider that the protein is precipitated after 
>extraction of integral membrane proteins with 1% Triton and eluted from 
>the antibody at high PH and exchanging triton with octylglucosyde, 
>always at physiologic pH.
>If anybody could help me in get rid of this fastidious contaminant, I 
>would be very grateful.
>Cordially,
>Giorgio Spagnol

	Since both of the contaminants are of lower molecular weight than
your protein of interest, could they be degradation products?

	How are you detecting proteins in your immunoprecipitation gel?
Are you radio-labeling?  Could your monoclonal antibody be getting labeled
somehow?  (Two polypeptide chains, right...)

	Alternately, could these contaminants just be tightly-bound co-
precipitants?  They might manage to cling on through the Triton and octyl-
glucoside, but dissociate during the SDS-PAGE.

	Sorry, I don't have any ideas about how to purify your protein, 
but depending on what these "contaminants" are, you would consider different
approcahes.

-- 
Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989  (Ph.D. perhaps 1998???)
Location  : Stanford University, Dept. of Structural Biology, Fairchild D-105
Keywords  : immunology, music, running, Green



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