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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: "Nadir T {- dot -} Mrabet" Nadir {- dot -} Mrabet {- at -} MEDECINE {- dot -} UHP-NANCY {- dot -} FR Date: 2010-05-19 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 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 > >> 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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