Wessel-Flugge Method for Protein Precipitation

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

In article <[Only registered users see links. ].net> , Jeremy Kamil <[Only registered users see links. ]> wrote:

I don't know, it always worked extremely well for me. Beats TCA any
day. I always remove the aqueous layer as completely as I can -
basically, until the meniscus collapses. I also always spin at all steps
much harder than, IIRC, indicated (1 min at 12K). The protein is on
interphase, so it is not a good idea to remove it. Don't think it's
detergents causing it in your case, since I used to remove 1%
Triton X-100 with no problems. Maybe try using more chloroform
during the initial denaturing step?

DK

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: