Wessel-Flugge Method for Protein Precipitation

Dale Beach via methods%40net.bio.net (by dbeach from email.unc.edu)
Tue Oct 31 01:39:34 EST 2006


jeremy-

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?

dale


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.
> 
> thanks,
> 
> Jeremy
> 
> 
> 


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