Not quite correct, look into Blue Native PAGE. There you can seperate
natively by mass.
Jürgen
..
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street,
Maia speaks about native PAGE for which protein mobility (migration)
depends on 3 different parameters as she states: charge, mass and shape.
Blue native PAGE, which might be the answer to Jacob's question, is a 2D
gel: Native in the first direction, then SDS-PAGE in the second one.
You
Dear Jacob,
I know that this is not the answer you were seeking, but for a modest
increase in the amount of protein required, a couple of analytical
ultracentrifugation experiments would be able to determine
stoichiometry and binding affinity for such a system. AUC has the
added benefit of being
You don't necessarily need the second dimension.
BN-PAGE gel:
protein A alone| some proteins you know the size as reference|protein B alone|
your mixture
You will be able to see in the mixture a) one or b) multiple bands, since the
Coomassie is equally distributed and attached to your protein
Thanks Jürgen.
Yes, you may show dissociation.
However, especially if you deal with assembly, then it might be
difficult, if not impossible, to tell your exact subunit composition if
you runBNP only in the first direction. Jacob mentions the possible
occurrence of complex assemblies (AB, BB,
Dear Jacob,
somewhat adding to list of 'not really answering your question', here is
the reference for native page that still uses a dye thereby trying to
limit the influence of the charge on the speed. Might be helpful as they
discuss some applications. Otherwise AUC really sounds like the
That's interesting. Thanks.
Maia
Nadir T. Mrabet wrote:
Maia speaks about native PAGE for which protein mobility (migration)
depends on 3 different parameters as she states: charge, mass and shape.
Blue native PAGE, which might be the answer to Jacob's question, is a
2D gel: Native in the
Subject: Re: [ccp4bb] Native Gel Theory and Practice
Here's just one example, which I quickly found from
Reisinger and Eichacker. Isolation of membrane protein complexes by blue native
electrophoresis. Methods Mol Biol (2008) vol. 424 pp. 423-31
Now Jacob has A 22 kDa B 17 kDa, the charge can
Dear Crystallographers,
I am trying to optimize a native gel experiment of a two-protein complex,
running the smallest-detectable amount of protein component A with varying
amounts of component B.
MWCharge MW/Charge
A 22 -5-4308
B 17-24 -702
This
Dear Jacob, I offer you my opinion.
Are you talking about electrophoresis? As far as I know it does not work
for the mass. The velocity of a protein depends on the charge at a
particular pH, the mass and shape of molecules etc. It's very difficult
to take all these things into consideration.
Dear all,
I am wondering if anyone is working on Blue Natve PAGE or other alternate
methods. I need some help to troubleshoot my Native PAGE experiments. It
will be great if anyone can help me in this regard. I am working on membrane
proteins which have pI around less than 7. i need to run
-
From: KN kn...@auckland.ac.nz
To: CCP4BB@JISCMAIL.AC.UK
Sent: Thursday, February 12, 2009 10:30 PM
Subject: [ccp4bb] native gel
Dear all,
I am wondering if anyone is working on Blue Natve PAGE or other alternate
methods. I need some help to troubleshoot my Native PAGE experiments
Hello Jacob,
The problem for native gel is that it is much more sensitive to a
single charge difference than size differences. Also, the gel pattern
may change greatly if you use a different buffer system. I had a case
2 years ago that my protein ran at 5 positions on a Laemmli(pH 8.8)
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