Hello Lisa,
I assume you have used NUPAGE gel from Novex in the second dimension. In
this case your streak has nothing to do with the rest of your experiment but
is only due to the type of gel. Something in it becomes stained in a very
strange way. I have observed this earlier and assume this has something to
do with the ampholines in the gel, but I have no proof for that. Only way so
far is to use a normal TriGlycine gel. People at Nvex in Germany know the
problem aswell but had no solution for that 2 years ago. Maybe someone can
fiddle that out with Novex.
Hope this helps a bit
Fritz Schweikart, PhD
_____________________________________________
Astrazeneca Biotech Laboratory
building 841, S-151 85 S–dert”lje, Sweden
fritz.schweikart@astrazeneca.com
-----Original Message-----
From: lisa bibbs [mailto:bibbs@scripps.edu]
Sent: den 11 april 2001 07:04
To: Recipients of ABRF List
Subject: 2D:need help with Amersham IPG strips
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!
This archive was generated by hypermail 2b29 : Tue May 01 2001 - 14:07:05 EDT