Protocol for urea gel electrophoresis to seperate proteins

[Madden, Robin]

Paul -

I came across your name when searching for methods to separate proteins
in urea. (see below) In the short protocol, you don't mention the
running buffer used.   Is it simply a regular Tris/SDS running buffer
with urea added to it?

Thank you for your help.

 

Robin Madden

 


Protocol for urea gel electrophoresis to seperate proteins


Paul S. Brookes. brookes at uab.edu
<mailto:methods%40iubio.bio.indiana.edu?Subject=Protocol%20for%20urea%20
gel%20electrophoresis%20to%20seperate%20proteins&In-Reply-To=> 
Fri Jul 9 14:03:47 EST 1999 

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Here's what I use for a 13% mini gel (Biorad).  You'll have to work out
for
yourself the amount of polyacrylamide to get 10%.
 
                                Separating Gel:         Stacker:
10% SDS                         80ul                    30ul
Protogel                        3.47ml                  0.72ml
Urea                            2.88g                   1.08g
APS (100mg/ml, fresh)           80ul                    24ul
Buffer                  1.6ml                   0.3ml
TEMED                   4ul                     2.4ul
Water                           2.8ml                   1.82ml
Total                           8ml                     3ml
 
Buffers are:    Stacker: 3.8% w/v Tris in H2O, pH6.7
                Separator 11.3% w/v Tris in H2O, pH 8.9
 
Protogel (30% w/v acrylamide/bis-acrylamide) is from National
Diagnostics.
 
Also, include Urea in your sample buffer (8M) and no glycerol.  It
should be
dense enough to sink to the bottom of the wells like this.  I usually
electrophorese for 1.5hrs at 85V (but that will depend on the MW of your
protein of interest).
 
Regards
PSB