native agarose electrophoresis of megadalton protein
Paul S. Brookes.
brookes at uab.edu
Wed Aug 30 08:44:06 EST 2000
A. J. Bowling (abowling at mail.utexas.edu) wrote:-
"has anyone out there had any experience and/or luck separating large
(megadalton) native protein complexes on agarose gels? in fact, any info on
electrophoresing proteins in agarose gels would be useful (ie TAE buffer or
normal acrylamide buffers, or something else?) im having trouble getting the
coomassie out of the gel after staining."
Not agarose, but polyacrylamide. The ref' that I use for mitochondrial
respiratory complexes is Schagger & Von Jagow (1991) Anal. Biochem. 199,
223-31. A 5-15% gradient gel gives good separation from 880kD (complex I)
to 140kD (complex II). Destain in 25% methanol, 10% acetic acid, 1-2hrs
room temp'. To speed this up try putting a rolled-up clean tissue in the
tray to absorb stain.
You can also microwave it on high for 20s. Repeat 3-4 times with fresh
destain buffer and the whole process is done in 5 minutes. However this
depends on what you want to do next - if you want intact complexes for
enzymatic analysis then too bad. If you're just wanting to western blot or
do a 2nd dimension then denaturing isn't a problem, so microwave it.
Dr. Paul S. Brookes. (brookes at uab.edu)
UAB Department of Pathology, G004 Volker Hall
1670 University Blvd., Birmingham AL 35294 USA
Tel (001) 205 934 1915 Fax (001) 205 934 1775
The quality of e-mails can go down as well as up
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