we get around this problem by running the DTT (dithiothreitol, Fisher #
BP172-5) denatured protein lysate on a gradient gel 7.5 % - 12 %. BME has
been know to be problematic in differential reducing gels in our hands.Good
luck, Donna Montague, M.S.
Research Associate
Physiology/Biophysics and Orthopaedic Surgery
University of Arkansas for Medical Sciences
(501) 603-1239


-----Original Message-----
From: Todd Miller [mailto:tmiller103@hotmail.com]
Sent: Thursday, October 11, 2001 9:36 AM
To: histonet@pathology.swmed.edu
Subject: collagen SDS-PAGE


Hi all.  I was wondering if anyone has a protocol for doing collagen gels.  
I've been running 5% non reducing SDS-PAGE gels to separate type I and III 
collagen from muscle tissue.  I've been using a delayed reduction technique 
where the dye front is run about 1/3 of the way into the separating gel, 
then the power is turned off and BME added to the wells to allow the type 
III collagen to migrate into the gel.  My gels look great, except the type 
III doesn't appear to be affected at all by the BME.  The alpha and beta 
chains run great, but there is a band right up near the wells that I'm 
assuming is the type III.  There is no visible difference between the 
reduced and non-reduced lanes.  Is it possible for BME to go bad?  I made 
everything myself, so I know there were no reducing agents in any of the 
buffers etc...  Has anyone else had this problem or have any suggestions?  
Thanks.

Todd Miller, Ph.D.
University of Pennsylvania School of Medicine
Department of Microbiology
211 Johnson Pavilion
3610 Hamilton Walk
Philadelphia, PA 19104-6076

Phone: 215.898.6501
Fax: 215.898.9557
email: tmiller2@mail.med.upenn.edu


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