Wessel-Flugge Method for Protein Precipitation
(by dbeach from email.unc.edu)
Tue Oct 31 01:39:34 EST 2006
I've been using TCA ppt prior to MS as well. Could you please send
along the Wessel-Flugge protocol or a complete reference?
Our MS facility has recommended a straight acetone ppt. Any reason not
to use this method?
Jeremy Kamil wrote:
> Hi, I'm trying to concentrate dilute protein mixtures in my tandem
> affinity purification eluates for mass spec analysis. Specifically, I
> need to get rid of the EDTA and EGTA as well as the 0.1% tween or NP-40
> that is in my elution buffer.
> I saw that some people use the Wessel-Flugge method (Anal Biochem 1974)
> for this application (Methanol, Chloroform precipitation). However, in
> my hands I found it didn't seem to work great, at least I don't see a
> nice pellet.
> I've had some success in the past with TCA but often end up with some
> residual acidity even after many acetone washes. Anyway, at least with
> TCA I got a nice pellet every time.
> My question re: Wessel-Flugge is, HOW MUCH OF THE UPPER LAYER ARE YOU
> SUPPOSED TO REMOVE??? Should I get rid of the upper phase all the way
> down to the chloroform layer, or leave a bit above of the chloroform??
> The original publication says to remove the upper layer above the
> chloroform and interphase. But I don't see much of an interphase, except
> in some samples where I saw a diffuse band of white above the chloroform
> layer on the bottom. so, in case that diffuse white stuff was my
> protein, I left maybe 150 uL on top of the chloroform. In the end I did
> not see much, if any, precipitate and was pretty disappointed since the
> method seemed so promising.
> any advice would be very much appreciated. I found the description by
> Wessel and Flugge pretty cursory.
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