R.Jayakumar at RoswellPark.org
Thu Mar 4 12:32:21 EST 2004
I have been doing lots of SDS-PAGEs for the past several years and I have a few ideas about what is going wrong. It will be usefull if I know the following additional information:
1) The voltage conditions the gels are being run and the time usually taken to finish the run at those voltage conditions.
2) Is the gel being cooled or is it being run at room temperature? If cooled, at what temperature is the cooling set to?
3) How long do you leave the gel (running and stacking) for polymerization?
4) What are the chemical components that you have not changed so far.
5) What is the sample buffer composition of your 1X laemmli buffer (especially the reducing agent being used and its 1X concentration).
6) What is the thickness of the gel?
7) Siliconized plates are not used for protein separations. But companies do sell siliconized glass plates for DNA work. But it would make the issue clearer to verify if the plates are siliconized. If so, use ordinary plates. But I dont think that is the cause of the problem. But it will help in narrowing the possible causes.
The answers would be helpful in finding a cause for your problem.
best of luck
> From: owner-methods at hgmp.mrc.ac.uk on behalf of Steve Nothwehr
> Sent: March 4, 2004 10:27 AM
> To: methods at hgmp.mrc.ac.uk
> Subject: SDS-PAGE problem
> Hello All,
> For the last few weeks we have been experiencing a problem with our
> SDS-PAGE gel system (a typical vertical gel system from CBS scientific
> for running 18 cm long gels). What happens is that for the first part
> of the run all looks well and we see a nice tight bromophenol blue dye
> front and good separation of our pre-stained standards. Then about
> half-way through the run the dye front becomes more diffuse and
> streaky. Also we observe the gel slipping down between the plates in
> the direction of the bottom buffer reservoir. When we detect the
> proteins from such gels the bands appear streaky and diffuse as if
> there was a non-uniform electrical field through the gel. Generally,
> the plates behave a bit like they have been siliconized (even though
> we have not knowingly treated them with anything out of the
> We doubt if the gel reagents are a problem because we use the same
> reagents on our mini SDS-PAGE gel system and we never experience any
> problems. Almost all of the reagents have been re-made since we began
> experiencing the problem. In addition, the problem with our large
> SDS-PAGE system is intermittent. This made me suspect the plates. We
> typically wash our plates in warm sudsy water, rinse with distilled
> water, drip dry, and wipe down with 95% ethanol with a kim-wipe. We
> tried soaking our gel plates in 2 M NaOH for 2 hr followed by washing
> to get rid of any foreign substances but this didn't solve the
> problem. Recently we bought all new gel plates and for a while the
> problem seemed solved but just yesterday we had a gel run poorly once
> again. The problem doesn't seem to correlate with using a particular
> rig or position on the rig (each rig has 2 positions).
> I have been running SDS gels for 20 years (9 years with the CBS
> system) and have never experienced a problem like this. We are
> desperate. If you have any ideas please let me know. Thanks.
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