Ming,
On that note, here is a Blue Native PAGE protocol that reportedly
improves the separation of basic proteins. A friend and I have used it
for native protein separation, but not necessarily for basic proteins.
Word of warning: don't use histidine salts or Tris-HCl in the cathode
buffer. The Figure 1 caption mentions this buffer shouldn't have
chloride ions in it.
Regards,
Steve Darnell
Electrophoresis. 2006 Oct;27(20):3949-51.
Discontinuous native protein gel electrophoresis.
Niepmann M, Zheng J.
Institute of Biochemistry, Justus-Liebig-University Giessen, Giessen,
Germany.
[EMAIL PROTECTED]
Analysis of the oligomeric state of a native protein usually requires
analytical
ultracentrifugation or repeated gel filtration to calculate the
protein's size.
We have developed a discontinuous native protein gel electrophoresis
system that
allows the separation of even basic proteins according to their size,
oligomeric
state, and shape. This gel system combines the addition of negative
charges to
the proteins by Serva Blue G with a discontinuous buffer system and
gradient
gels. As in SDS-PAGE, chloride constitutes the high mobility anion in
the gel and
anode buffer. However, for sample focusing this system employs
histidine instead
of glycine as the slow dipolar ion following from the cathode buffer
to improve
migration of basic proteins. In addition, proteins run into gel pores
corresponding to their size and shape in the gradient gel. Using this
gel system,
we show that the polypyrimidine tract-binding protein (PTB) is a monomer.
PMID: 16991206 [PubMed - indexed for MEDLINE]
--
Steven Darnell
Department of Biochemistry
University of Wisconsin-Madison
Madison, WI USA
[EMAIL PROTECTED] said the following on 8/26/08 12:20 PM:
You could try Coomassie Blue Native gel. It's a very neat technique and it
worked for me on a couple of occasions. In one unfortunate case, it
resulted in dissociation of a heterotetrameric complex, though.
Artem
Dear CCP4 community,
Sorry for the off-topic subject, but I would really appreciate some
suggestions and/or protocols relating to native gel electrophoresis of
basic proteins. I have used a general acidic PAGE protocol for my protein,
which has a PI of 9.5. Briefly, the protein was loaded onto a native gel
(I have tried both the pre-made Biorad gels (7.5% and a gradient gel:
4-15%) and freshly prepared Tris native gels adjusted to pH 6.8) and run
in a 1X acetic acid/b-alanine pH 4.5 running buffer. The electrodes were
reversed and the gel run on ice for ~ 2hrs at 100V. In all cases, the
native protein was unable to enter the gel. Some protein samples incubated
with heavy atoms were able to enter the gel (possibly indicating binding)
but these samples too had problems entering the gel as the bands were at
or just a little bit below the edge of the well. Any suggestions and
comments would be most welcome!
Thank you so much in advance for your help,
Sincerely,
Ming Lye
