Dear Jacob, I know that this is not the answer you were seeking, but for a modest increase in the amount of protein required, a couple of analytical ultracentrifugation experiments would be able to determine stoichiometry and binding affinity for such a system. AUC has the added benefit of being a solution phase system, and you can run it in the buffer of your choice.
If your system is as complex as you describe, then native PAGE *might* not unambiguously give you the answers you seek. HTH, David. ============================ David C. Briggs PhD Father, Structural Biologist and Sceptic ============================ University of Manchester E-mail: david.c.bri...@manchester.ac.uk ============================ http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB ============================ On 18 May 2010 21:04, Jacob Keller <j-kell...@md.northwestern.edu> 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 > ******************************************* >
