SDS-PAGE and guanidine....

L.D. lnd at mail.utexas.edu
Fri Jun 18 14:57:50 EST 1999


Dima Klenchin wrote:

> In article <376916DD.462E1B35 at mail.utexas.edu>, lnd at mail.utexas.edu wrote:
> :"S. Findley" wrote:
> :
> :> Can you exchange the Guanidine for Urea using Those Amicon
> :> Microconcentrators? (or something similar)
> :> --
> :> Seth D. Findley               polliwog at u.washington.edu
> :> Department of Biochemistry    Phone: 206-543-1710
> :> Health Sciences Building J579
> :> University of Washington, Box 357350, Seattle, WA 98195
> :
> :That's realy good idea and works perfectly. Use green centricone tubes
> :(Amicon) and centrifuge your sample with shared new buffer, at 500-100
> :rpm (Sorval, HB-4). It takes time, but you can get your sample not only
> :washed from Guan..  but also concentrated.  I use this way to
> :concentrate protein(s) el.eluted from SDS-PAGE. Sometimes I even use
> :those tubes for el.elution, set directly into rod PAGE equipment.
> :
>
> That's really bad idea because it is even more tedious than methanol/
> chloroform pption he does not want to use. It is also less quantitative
> and MUCH more expensive. I'd say, dilute guanidinium to ~ 1 M and
> do TCA. If protein concentration under such dilution becomes too
> low for TCA, I'd stick with MeOH/Choroform - it's not really much more
> work than TCA.
>
>         - Dima

I understand that one may prefer playing with alcohols (just to precipitate
stuff) :),  but I doubt that's so bad idea using centricone tubes compared
with TCA and/or Chlor/MetOH precipitation. All depends what proteins are you
working with and how much of them you have.  Many transmembrane proteins,
especially those being highly glycosilated or modified, do not tend to be
precipitated well by those methods. In addition, one has to consider the
problem of recovery. In this light, centricone tubes are just perfect.

L.D.




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