Wessel-Flugge Method for Protein Precipitation

[Jeremy Kamil]

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