mthomas at hsc.usf.edu
Tue Apr 18 12:08:03 EST 2000
In article <Pine.SGI.4.10A.B3.10004141104470.758500-100000 at umbc7.umbc.edu>,
Elizabeth Rogers <erogers at umbc.edu> wrote:
>Any tricks to running IEF gels? We have Biorad IEF gels and want to
>separate 2 proteins that run together on SDS-PAGE for N-terminal
>sequencing. Our first stab didn't do too well at the high voltage (top
>buffer leaking into bottom buffer, fried gel..) Do we need to fix the
>gel or get rid of ampholytes before electroblotting to PVDF? The info we
>have is pretty skimpy and mostly related to 2D gel applications and/or
>standard fixing and staining. Thanks
>Elizabeth Rogers Department of Biological Sciences
>Phone:(410) 455-2285 University of Maryland Baltimore County
I too tried bio rad IEF gels and found pouring my own worked better. I used
the protocol in "current protocols in protein science" The protein version
of the red book.
I used pharmacia's pharmalytes (not too expensive and fairly reproducable)
I found that putting some vaccum grease on the gel gasket before I assembly
the rig helped with leaking between the uper and lower tanks (this can be a
major problem). Makes for a pain to clean up but the pH of the top and
bottom stay what they are supposed to be!
To do a western on an IEF gel you need to wash them in 1% SDS, 50% methanol,
5mM Tris pH 7.9. I would wash them 5 times for 10 minutes in about 100mls
or so with shaking. Then blot them as normal (normal for me is overnight at
90mA with PVDF membranes). This to is in the current protocols book. They
also have tips on how to do flourography (sp?) with tritium labled proteins
in IEF gels.
I also found that silver staining works much beter than coomasie as coomasie
will stain some of the ampholytes. You can wash them out than stain, but by
then you could have silver stained it and be done. The silver stain
protocol in the red book (current protocols) worked best. I tried bio rad's
silver kit and wasn't impressed. Much better to do it with home made
solutions. I also found you had to load a fair amount of protein to see
them in the IEF gels compared to a normal SDS gel. Something that would be
overloaded in SDS looks about right in IEF.
I also found if I ran 10cm gels I could get fairly good resolution and this
saved on ampholytes and you only had to run it about 3 hours at 5W instead
I'm sure I'm missing something but that is most of my IEF gel secrets. I
also found when I tried to find protocols about them that most people were
doing IEF tube gels for 2D and that there seems to be some significant
tricks that work for those but not normal IEF slab gels.
Hope this helps.
Matthew Thomas, Ph. D
H. Lee Moffitt Cancer Center, University of South Florida
mthomas at hsc.usf.edu
"I refrain from publishing for fear that disputes and
controversies may be raised against me by ignoramuses."
Sir Isaac Newton, correspondence to Liebniz
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