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 > > > > 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> 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 >>>> ******************************************* >>>> >>>> >> - 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/
