[Protein-analysis] Re: What is interfering with SDS-PAGE?

Duncan Clark blackhole at abuse.plus.com
Fri May 26 04:36:33 EST 2006


Historians believe that in newspost 
<mailman.1085.1148492571.16885.proteins at net.bio.net> on Wed, 24 May 
2006, Rafael Garcia <rgarcia at errc.ars.usda.gov> penned the following 
literary masterpiece:
>I am solubilizing difficult proteins using a pretty harsh extraction solution:
>7M Urea
>2M Thiourea
>0.05 M DTT
>0.01M Tris
>2.5% SDS
>1% N-lauroylsarcosine (detergent)
>protease inbitor cocktail
>pH7.4
>
>Something in this mixture makes SDS-PAGE gels look streaky and dark.  If I clean up and concentrate the extracted protein with ultrafiltration, the
>gels work much better.  (dialysis followed by lyophilization doesn't seem to work).  Which component in this extraction solution is causing the
>problem?

Remove one component at a time, add the remainder to any protein and 
load into a single lane of a gel. Repeat removing another component etc. 
Identify which component causes the problem.

Simple fault finding experiment.

Duncan
-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.


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