SDS-PAGE problem

Jayakumar, R R.Jayakumar at RoswellPark.org
Sun Mar 7 11:41:27 EST 2004


Dear Steve
     sorry for this late reply.  Anyway, I carefully went over your letter (attached below).  It looks like you are doing everything right.  So the only possible cause might be one which normally a lot of people overlook, since we are biologists and not electricians.  Did you check the resistance factor in the electric flow between your powerpac 3000 (which is optimized for BioRad's protean II and their other products), and your CBS electrophoresis unit.   Since you tell me that you run your gel at room temperature, there might a good possibility of high resistance in the circuitary building up localized heating (the most likely reason for protein and dye streaking) towards the later parts of the run.  this might explain why your gel looks OK when the run is terminated very early and the gel sags when it is run for longer times.  The only other reason (other than problems like. very low percent <7% gels, poorly secured gel plates and insufficient pressure holding the gel plates together - which I believe does not happen with you), I could think of for the sagging of the gel, is softening of the polyacrylamide gels due to heating up of the gel.   
    We also use the same Powerpac 3000 in our labs for running  large 7% SDS-PAGE gels with our BioRad protean II unit, at 25mA constant for 5-6 hours under cooling conditions of 15C (lower than that and you might get SDS problems).  We have never encountered those problems which you desribe, probably since we cool the gels.  BioRad recommends cooling of these gels. 
      I suggest trying again, but this time cool the gels to 15C, increase the current to 25mA and finish the run quickly.  Note the starting voltage and ending voltage and the total time taken.  I can cross check your start and end voltages with mine and come to an idea about what is going on.  Take care to cool the gel (with buffer) for atleast 15-30 minutes till the temperature of the buffer and gel has reached operating temperatures of 15C before loading protein samples.  I think that should solve your problem.
      bye and best of luck.  Please keep me updated on your problem.

Jai



> From: 	Steve Nothwehr
> Sent: 	March 4, 2004 2:00 PM
> To: 	Jayakumar, R
> Subject: 	Re: SDS-PAGE problem
> 
> <<File: SDS-PAGE sample buffer.DOC>>
> Jai,
> 
> Thanks for offering to help.
> 
> > 
> > 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.
> 
> We often run them at 5 mA constant for ~ 16 hr at 12 mA constant for 6-8 hr.
> We have had the problem occur under both scenarios.
> 
> 
> >   2) Is the gel being cooled or is it being run at room temperature?  If
> > cooled, at what temperature is the cooling set to?
> 
> The gel is run at room temperature and because the gels are run so slowly we
> do not notice any heat build-up.
> 
> >   3) How long do  you leave the gel (running and stacking) for polymerization?
> 
> We typically allow the resolving gel to polymerize from 45 min to 2 hr and
> the stacker to polymerize for 45 min to 2 hr also.
> 
> >   4) What are the chemical components that you have not changed so far.
> 
> We have made new Tris, SDS, and are using a fresh bottle of monomer/bis
> acrylamide from Bio-Rad.  I'm not sure if we have recently changed the TEMED
> or ammonium persulfate but we haven't noticed any polymerization problems.
> 
> >   5) What is the sample buffer composition of your 1X laemmli buffer
> > (especially the reducing agent being used and its 1X concentration).
> 
> I have attached a protocol for making sample buffer.  We usually use it at
> 2X strength.
> 
> 
> >   6) What is the thickness of the gel?> 
> 
> 0.75 mm
> 
> >   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.
> >   
> 
> We were using plates from CBS scientific and they assured us that the plates
> were not treated with anything including silicon.  We then had plates made
> by a local glass shop.  Again the glass was not treated with anything.  Both
> sets of plates exhibit the problem.
> 
> 
> > The answers would be helpful in finding a cause for your problem.
> > best of luck
> > Jai
> > 
> 
> I should emphasize that all of our protocols are the same as we have used
> for many years and the problem revealed itself just in the last couple
> months.
> 
> This morning we had a new idea about where the problem could come from.  We
> are pretty sure that we only observe the problem when we use a certain power
> supply, a Bio-Rad  PowerPac 3000.  I just called Bio-Rad and they agreed
> that this was a possibility although there doesn't seem to be a history of
> such problems with this model.
> 
> Thanks again for any advice.
> 
> Steve
> 
> 
> 
> 
> 
> > 
> > 
> >> ----------
> >> 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
> >> ordinary).
> >> 
> >> 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.
> >> 
> >> Steve
> >> 
> >> 
> 
> -- 
> Steven Nothwehr, Ph. D.
> Associate Professor
> Division of Biological Sciences
> 401 Tucker Hall
> University of Missouri
> Columbia, MO 65211
> 
> Ph: 573-884-6461
> Fax: 573-882-0123
> 
> E-mail: nothwehrs at missouri.edu
> 
> 
> 
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