How about using static light scattering to determine the actual molecular weight or dynamic light scattering to measure the diameter of the complex.
Sheemei ________________________________ From: Jürgen Bosch <jubo...@jhsph.edu> To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, 19 May 2010 11:00:24 Subject: Re: [ccp4bb] Native Gel Theory and Practice 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 <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/
