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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 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 be disregarded in BN PAGE. If we do the math for all the theoretical complexes and assume globular shape for all of them. 78 AABB (22+22+17+17) 61 AAB (22+22+17) 56 ABB (22+17+17) 39 AB (22+17) 22 A 17 B I'd use a higher percentage gel 10-20% then you should be able to separate the 6 species mentioned above. Jürgen P.S. just trying to be helpful On May 19, 2010, at 11:40 AM, Maia Cherney wrote: > Yes, you can separate by electrophoresis, that's why we use it, but we > cannot calculate accurate mass of complexes. > Maia > > > 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 >>>> ******************************************* >>>> >>>> >> >> - 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/ CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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