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, ABB, AAB).
Nadir
Pr. Nadir T. Mrabet
Structural& Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet<at> medecine.uhp-nancy.fr
On 19/05/2010 16:05, Jürgen Bosch wrote:
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 in a non-denaturing way it is directly proportional to the
molecular mass of your protein complex.
I'm searching for the protocol which was I believe either from the
Görg group or a group in Berlin, perhaps also Mann demonstrating
dissociation of a multimeric complex in dependence of reducing agent.
If I find it I will post it tonight.
Jürgen
On May 19, 2010, at 10:01 AM, 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 first direction, then SDS-PAGE in the second one.
You actually need both data to infer stoechiometry and subunit
composition.
Nadir
Pr. Nadir T. Mrabet
Structural& Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet<at> medecine.uhp-nancy.fr
<http://medecine.uhp-nancy.fr>
On 19/05/2010 13:01, Jürgen Bosch wrote:
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, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-3655
http://web.mac.com/bosch_lab/
On May 19, 2010, at 1:31, Maia Cherney <ch...@ualberta.ca
<mailto:ch...@ualberta.ca>> wrote:
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. Otherwise this would be a
very convenient method, much easier than the analytical centrifugation
or gel-filtration that are usually used. However, electrophoresis
does
not work for mass determination. Besides, complex formation hugely
depends on the protein concentration. If you dilute your mixture, your
complexes might dissociate. There is equilibrium constant between
different types of complexes.
Maia
Jacob Keller wrote:
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.
MW Charge MW/Charge
A 22 -5 -4308
B 17 -24 -702
This experiment is partly to determine stoichiometry, but also to
determine roughly the strength of the interaction.
B definitely runs much faster than A alone, as predicted, but I am
wondering what to expect with various oligomers. Should ABB run faster
or slower than AB? What about AABB? Theoretically, AA should certainly
run slower than A, and BB slower than B, simply because the
mass/charge ratio is the same, but the overall mass is greater. But
what happens when you have AAB, for example? There must be an equation
relating the mass/charge and mass (and perhaps gel percentage) to the
speed traveled in the gel--but what is the equation?
Thanks for your consideration,
Jacob
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: j-kell...@northwestern.edu <mailto:j-kell...@northwestern.edu>
*******************************************
-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-3655
http://web.mac.com/bosch_lab/ <http://web.me.com/bosch_lab/>
