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Protocol for urea gel electrophoresis to seperate proteins

Protocol for urea gel electrophoresis to seperate proteins - Protocols and Methods Forum

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Old 12-19-2006, 05:55 PM
Madden, Robin
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Default Protocol for urea gel electrophoresis to seperate proteins



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
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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






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