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<i>A. J. Bowling (abowling@mail.utexas.edu) wrote:-<br>
<br>
</i>"has anyone out there had any experience and/or luck separating
large <br>
(megadalton) native protein complexes on agarose gels? in fact, any info
on <br>
electrophoresing proteins in agarose gels would be useful (ie TAE buffer
or <br>
normal acrylamide buffers, or something else?) im having trouble getting
the <br>
coomassie out of the gel after staining."<br>
<br>
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.<br>
<br>
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.<br>
<br>
Regards<br>
PSB<br>
<br>
<br>
<font color="#000080">_________________________________________<br>
</font><font color="#FF0000"><b>Dr. Paul S.
Brookes.</b>
(brookes@uab.edu)<br>
</font><font color="#000080">UAB Department of Pathology,
G004 Volker Hall<br>
1670 University Blvd., Birmingham AL 35294 USA<br>
Tel (001) 205 934 1915 Fax (001) 205 934
1775<br>
<a href="http://peir.path.uab.edu/brookes" eudora="autourl">http://peir.path.uab.edu/brookes</a><br>
<br>
<b>The quality of e-mails can go down as well as up<br>
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