Protein Precipitation: how about microconcentrators?

Basavaraju Shankarappa bsh at MED.PITT.EDU
Mon Apr 18 09:20:09 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...)

> 	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
> 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              /
> 

	How about all those micro concentrators from amicon and millipore.
Although I tend to believe that you lose a lot of sample in this method,
may be it is worthwhile and quick enough for you to try it out.  They have
a whole sleuth of cut off sizes for this.  
Raj Shankarappa
bsh at med.pitt.edu




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