What is interfering with SDS-PAGE?

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?

Rafael Garcia
Chemical Engineer
USDA-ARS
Eastern Regional Research Center
600 East Mermaid Lane
Wyndmoor PA 19038
voice: 215-836-3743
fax: 215-233-6795
[Only registered users see links. ]

Rafael Garcia <[Only registered users see links. ].usda.gov> wrote:


Horrible mixture (typing errors?). A lot of things, particularly
denaturing salts and virtually no buffering (10 mM Tris??). Does a
standard SDS-PAGE really give you anyhing at all? (gel System?)

(meaning what?)


Not enough info.

I would look at the Urea first. Surely there is a reason why you have
it there. But 7M? You are boiling things in SDS - 2-mercapto anyway, is
it that resistant to denaturation? (I know it happens, but these are
extreme conditions).

--
Kaj

Historians believe that in newspost
<[Only registered users see links. ].ne t> on Wed, 24 May
2006, Rafael Garcia <[Only registered users see links. ].usda.gov> penned the following
literary masterpiece:

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.

Rafael Garcia wrote:


Urea and thiourea can cause problems in SDS-PAGE, especially at high
concentrations. Also the presence of high concentrations of non-ionic
detergent can interfere with SDS-binding to the protein.

In addition, it might be better to solubilise at the pH of SDS-sample
buffer (6.8) instead of 7.4. You may be interfereing with the stacking
effect of DISK-electrophoresis (see the original Ornstein-paper from
1962 for a detailed explanation). This too would result in broad bands.

Are you boiling your sample before electrophoresis? This can interfere
with solubility, try 10 min at 60 degrees instead or even, since you are
working with urea, 30 min at 37 degrees.