Protein Precipitation

John McDougall johnmcd at fagmed.uit.no
Mon Apr 18 06:05:48 EST 1994


In article <2oejvv$scv at fermat.mayo.edu>, volcs at fermat.mayo.edu (Sam
Volchenboum) wrote:

> I need to precipitate a protein sample coming off a FPLC column for
> subsequent SDS-PAGE.  I was going to precipitate with 10% TCA and then wash
> with 10% TCA, but I read that the TCA can change the pH so as to affect the
> PAGE.  Then someone told me to wash with acetone.  Fine, but can't I just
> precipitate with one volume of acetone from the start?  Anyways, do any of
> you have ideas/protocols/refs which will precipitate small amounts of
> protein and not affect the PAGE? (Or should I just use TCA and be done with
> it...)
> 
> Thanks,
> Sam


Sam: 
	TCA or TCA/deoxycholate pption and then re-pHing with saturated un-pHed
Tris, once your sample is in loading buffer, so that the BPB is blue again
is one that I use often. However, it is easy to over pH your sample.
Another is to ppte your proteins with 5-10X vol acetone -I find 5 vol is
usually good enough but you can use 10 vol to be sure. A third way is, in
fact, a method for removal of lipids:
	Add 0.4 ml of methanol to 0.1 ml of protein soln. Vortex and add 0.1 ml
chloroform, vortex and centrifuge 1 min (microcentrifuge). Add 0.3 ml
water, vortex and centrifuge 1 min. Remove upper layer and discard. Add 0.3
ml methanol to the lower chloroform phase, mix and centrifuge 2 min. Remove
the super and dry the protein pellet.

Better late than never.
-- 
John McDougall       / TEL: 47-776-44479
NFH                  / FAX: 47-776-71832
Univ. of Tromsoe     / E-MAIL: johnmcd at fagmed.uit.no
N-9037 Tromsoe       /
 Norway              /



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