svetlov at oncology.wisc.edu
Wed Nov 25 10:51:46 EST 1998
In article <73fn8p$4uf at bellboy.ucc.uconn.edu>, "Qiang Zhang"
<qiz97001 at uconnvm.uconn.edu> wrote:
> hi, everyone, i have a question about Protein SDS-PAGE. i run the gel which
> is 0.75mm thickness, 2cm height of stacking get plus 12cm height of
> separating gel under constant current of 30mA for two gels(15mA for each).
> it runs so slowly that takes about more than 10 hours to run to the bottom.
> the time is much longer than that in my lab's protocol. the concentration of
> the stacking gel and separating gel are 3% and 10% respectively. i am sure
> that there is no error in my all buffer. is there anyone who has experience
> in it can give me some advice about why it is so slow and what i should
> check about it. how long do you spend to run the gel like this? thank you
> for your help and advice.
Depending on the buffer this gel should be done in under three hours max.
With Tris-Glycine buffer I'd expect this run to be done in under two
(although I run such gels a bit faster, at constant voltage of 110 which
means starting at 25 mA). Barring the instrumental error on your power
supply the only naturalistic explanation of this extremely long running
time is a problem with composition of your gels and/or your buffer. To find
out which if not both is the problem borrow ALL the solutions for making
the gel from somebody who does not have this problem (or use commercial
stocks) and a stock solution for the running buffer. If that does take care
of the problem, replace one of these at a time. If not, check your power
supply. Oh, before you do any of that sort check that your gel apparatus
does not leak or that intended current flow is not compromised in any other
way (by arching, broken electrode etc.). BTW, what were the voltmeter
readings during your runs and what buffer did you use?
That's about it. Despite my vast experience with SDS-PAGE and Might&Magic
series I can not recall any spell or curse that would adversely affect the
running time of the e/phoresis, so it has to be a factor that impacts one
or more of the electric/electrochemical parameters.
P.S. Don't you guys just love the questions like "I did everything right,
but it does not work. Tell me what could have happened other than something
is screwed up"?
Vladimir Svetlov, PhD
McArdle Lab for Cancer Res.
1400 University Ave.
Madison, WI 53706
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