SDS-PAGE problem

[Steve Nothwehr]

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.

Regarding the message above from Taskan suggesting that the ethanol
could be the problem: I am intrigued by this possibility.  The only
argument against this is that we also frequently run mini-protean gels
(Bio-Rad system) and we treat those plates with the same 95% ethanol
as with the larger plates and we have never had a problem with the
mini gels.  Nevertheless maybe the problem is only revealed after a
much longer run since the minigels are typically only run for 90 min.

Thanks again for any additional advice.

Steve