lisa,
what ampholyte conc are you using? it looks to me like the concentration is too high and this is causing the nasty back ground. i had the same problem a while back and lowered my pharmalyte conc to 0.5% from 2%. other folks in my lab have used as low as 0.4% and have had very nicely focused gels. give it a try.
jane
Jane A. Nagel, MSc
Genetics Institute
87 Cambridge Park Dr
Cambridge, MA 02140
617 665 8597
fax 617 665 8878
1 888 577 1500 x8597
>>> lisa bibbs <bibbs@scripps.edu> 04/10 5:21 PM >>>
Sorry I had to attach a picture so that you would know what we are
seeing. We have run several different strips and have remade all of
the solutions ...... Does anyone recognize this problem?
Below are the conditions that we have run. By the way, if we
transfer it, it doesn't show up.
If anyone can help, I would really appreciate it.
Thanks
Lisa
>
> >>
> >>I talked with you earlier on about a problem I am having with 2D.
> >>Attached is a gel picture of a 2D SDS PAGE STANDARD. My concern is the
> >>large streak across the gel. I ran the first dimension under the
> >>following conditions:
> >>
> >>For 1st Dimension:
> >>2D SDS PAGE STANDARD (7.5ul)
> >> DTT (18mM)
> >>Rehydration solution with IPG buffer pH 3-10L
> >>Immobiline Drystrip pH 3-10 L
> >>IPGphor Isoelectric focusing unit with following protocol
> >>
> >> 30V 12.00hrs
> >> 250V 1.00hrs
> >> 500V 1.00hrs
> >>1000V 1.00hrs
> >>8000V 10000Vhr
> >>
> >>For 2nd Dimension:
> >>XCell Mini-Cell electrophoresis system
> >>SDS Equilibration buffer with DTE
> >>12% Bis Tris 2D gel
> >>MOPS buffer
> >>Multimark Multi-Colored Standard
> >>Coomassie blue R 250 stain
> >>Destained with 50:50 Methanol and water
> >>
> >>
> >>
> >>I have tried the following and I still got this same streak.
> >>
> >>Freshly prepared rehydration solution with IPG buffer pH 3-10L
> >>Freshly prepared equilibration solution
> >>New IPG Cover Fluid (from Amersham)
> >>2 different batches of Immobiline Drystrip pH 3-10 L
> > Immobiline Drystrip pH 4-7L
> >>Very clean strip holders
> >>Freshly prepared coomassie blue R-250 staining solution
> >>
> >>The streak has been consistent in all of the gels that I ran for 2nd
> >>Dimension. It seems to start at pH 7 and moves diagonally towards the low
> >>molecular weight multimark standard at pH 3.
> >>
> >>
> >>I also tried running 1ST Dimension with rehydration solution alone (
> >>without 2D SDS PAGE STANDARD) and I got the same streak.
> >>
> >>NOTE! I ran 1ST Dimension on some samples for a customer. My customer ran
> >>2nd Dimension using his own gel, apparatus and staining solution and he
> >>got a very similar streak. So, I think the problem may be in the 1st
> >>Dimension and not in the 2nd dimension.I also transferred my gel onto a
> >>PVDF membrane and the streak didn't show up on that.
> >>
> >>I would really appreciate your help on this and please call or email me
>>if you have any further questions. Thankyou!
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