GuCl in SDS Page

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Sat Nov 23 08:06:28 EST 1996


In article <E19yoA.3r5 at fsa.bris.ac.uk>, bijgh at zeus.bris.ac.uk (Jared Head)
wrote:

> Hi,
> 
> Can anybody tell me a quick way of prepping samples of protein in 
> guanadinium chloride, so that they will run on SDS PAGE.  The GuCl seems 
> to precepitate when mixed with SDS.  I've only got small samples so dialysis 
> will be difficult.
> 
> Cheers,
> 
> Jared
> 
> -- 
> Jared Head     at the Department of Biochemistry, University of Bristol
> 
>   "Happily unaware of the disadvantages of being a terrible writer with 
>                               nothing to say..."


Try TCA precipitation of your samples. To avoid GuCl precipitation, it is
necessary to dilute the sample (with water) to less than 500mM GuCl before
addition of TCA. Then, add an equal volume of 10% TCA (w/v in water), mix
well, incubate on ice for 30min, centrifuge for 15min at full speed in a
desktop microcentrifuge, remove (and discard) supe, wash pellet in 1ml of
ice-cold acetone, recentrifuge, remove (and discard) supe, air dry pellet
for 15´ (acetone must evaporate COMPLETELY). Dissolve pellet in Laemmli
sample buffer. The solution often turns yellow because of residual TCA. Do
not try to neutralize with NaOH or unbuffered Tris (as often recommended),
as this will increase the salt concentration of your sample, so it won´t
run well on your gel. Usually the sample turns blue again upon application
to the gel (if it doesn´t, don´t mind). 

Often (especially when purifying denatured, His-tagged proteins), GuCl can
be replaced by urea in the last two steps of the purification. Urea does
not disturb your gel run, so you don´t have to precipitate your samples. I
use 8M urea to replace 6M GuCl.

Hope it helps,
Frank


PS: sometimes TCA pellets are hard (or impossible) to redissolve in
Laemmli buffer, especially when working with high amounts of protein
(>50ug). Try adding solid urea to the sample to dissolve. It usually
works....

-- 
Dr. Frank O. Fackelmayer
Division of Biology
University of Konstanz
D-78434 Konstanz
Germany



More information about the Methods mailing list