protein electrophoresis problem
Frank O. Fackelmayer
Frank.Fackelmayer at uni-konstanz.de
Tue Jul 25 09:23:49 EST 2000
several things could be the reason for the strange running of your gel.
* general gel related problems (buffer composition, acrylamide purity,
amount of starter...). Try running a gel with a commerical mol.wt.
marker set. They must produce clear bands if your gel system is ok.
* overloading of the gel. Try using decreasing amounts, say 1/3, 1/10,
1/30, 1/100 of the amount
you used. If the smear persists until the amount of protein falls below
detection limit, it is not overloading.
* solvents. If you have used organic solvents in any step of your sample
preparation, some may be left and ruin the migration. See next point for
removing them or avoid them next time if possible.
* contaminating molecules: DNA, RNA, glycogen, lipids, organic solvents.
Try purifying your samples by precipitation with MetOH/Chloroform
according to Wessel/Fluegge (1984), Anal.Biochem. 138,141-143; this
precipitation removes nucleic acids (shear by sonication before
precipitation if you use viscous total cell extracts), lipids, and
salts. Airdry pellet thoroughly before redissolving in sample buffer.
Hope this helps,
Kun Qian wrote:
> Hello everybody!
> I have a simple question, but it's hard for me.
> I run SDS-PAGE using the protein samples prepared by myself, and stain the
> gel with commassie blue after ecletrophoresis. The troublesome thing is that
> there is always smear on the gel according to the place of each lane. I
> don't know if this means the protein is degraded, or should the staining
> appear to have some major visible band .
> I am sorry for my poor description, but if you are interested in this
> problem, I can email you a picture of the gel.
> thanks a lot!!!
> Kun Qian
> > >
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