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I think intact mass-spec would be the most robust. But you should be able to distinguish a 2-fold stoichometry difference on SDS-PAGE, if you can separate the two bands. Run a 15 or 18% gel long enough and I think you should be able to get some separation between 16.1 and 16.9 kDa. Jason. -- Dr Jason Busby Laboratory of Structural Biology School of Biological Sciences The University of Auckland Thomas Building 110 3A Symonds St Private Bag 92019 Auckland Mail Centre Auckland New Zealand ph: +64 9 3737599 ext 83888 On 10/05/2016, at 9:11 AM, khaja faisal tarique <[log in to unmask]<mailto:[log in to unmask]>> wrote: Hello everyone I am working on two proteins (X and Y) which forms complex with each other. Their molecular weight is nearly identical to each other, say 16.1 and 16.9 kDa. On size exclusion chromatography a trimeric complex is obtained (something around 50 kDa). Now the question is whether the complex is 2X and one Y or 2Y one X ? what is the best way to determine the correct stoichiometry between them. Just to add more information these proteins lack any tryptophan residues. I request you to give some idea or suggest any experiment to determine their correct ratio of association. Regards Faisal