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

Protein Forum Protein Forum


What is interfering with SDS-PAGE?

What is interfering with SDS-PAGE? - Protein Forum

What is interfering with SDS-PAGE? - Protein Forum


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 05-24-2006, 04:08 PM
Rafael Garcia
Guest
 
Posts: n/a
Default 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. ]



Reply With Quote
  #2  
Old 05-25-2006, 05:55 AM
kaj.stenberg@helsinki.fi.invalid
Guest
 
Posts: n/a
Default What is interfering with SDS-PAGE?

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
Reply With Quote
  #3  
Old 05-26-2006, 09:36 AM
Duncan Clark
Guest
 
Posts: n/a
Default What is interfering with SDS-PAGE?

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.
Reply With Quote
  #4  
Old 06-02-2006, 03:20 PM
Dr Engelbert Buxbaum
Guest
 
Posts: n/a
Default What is interfering with SDS-PAGE?

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.
Reply With Quote
Reply

Tags
interfering , sdspage


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
Page gel Jayakumar, R Protocols and Methods Forum 0 04-27-2008 07:55 PM
SDS PAGE younes sadramehr Protocols and Methods Forum 2 12-05-2006 07:21 PM
Native PAGE question Protein Forum 1 05-09-2006 07:21 AM
Our RNAi and SiRNA Page is Now Open! admin RNAi and SiRNA Forum 0 01-03-2006 08:34 PM
FS: Old Physics-Related Books Oldbooks78 Physics Forum 0 08-27-2003 02:32 PM


All times are GMT. The time now is 09:16 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 0.13017 seconds with 16 queries