Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Wessel-Flugge Method for Protein Precipitation

Wessel-Flugge Method for Protein Precipitation - Protocols and Methods Forum

Wessel-Flugge Method for Protein Precipitation - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 10-24-2006, 06:29 AM
Jeremy Kamil
Guest
 
Posts: n/a
Default 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



Reply With Quote
  #2  
Old 10-25-2006, 02:06 AM
DK
Guest
 
Posts: n/a
Default Wessel-Flugge Method for Protein Precipitation

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

Reply With Quote
  #3  
Old 10-31-2006, 06:39 AM
Dale Beach
Guest
 
Posts: n/a
Default Wessel-Flugge Method for Protein Precipitation

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:
Reply With Quote
Reply

Tags
method , precipitation , protein , wesselflugge


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Scientific method (was Re: Fusion In Utah (again)) Bill Vajk Chemistry Forum 12 09-29-2003 08:30 PM


All times are GMT. The time now is 06:10 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12143 seconds with 16 queries