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Re: [ccp4bb] Native Gel Theory and Practice |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: Native Gel Theory and Practice From: Jürgen_Bosch jubosch {- at -} JHSPH {- dot -} EDU Date: 2010-05-19 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 > 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-keller2@northwestern.edu >> ******************************************* >> >> CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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