Hi Sanjeev, In addition to all the excellent suggestions (trying different buffers/salts, co-expression, concentrator, incubate and set up crystallization screening, checking SPR, fusion), I have a couple more suggestions:
1) related to your question about cross-linker: do you have experimental structures or homology models of one or both components? If so, you could try a couple of protein-protein docking programs (Haddock, etc) and analyze the interface for close contacts where you can introduce disulfides. 2) you can also try lysing the cells together and co-purifying from the beginning. 3) try reductive methylation on the incubated complex prior to setting up screens if you have numerous exposed lysines. Thanks, Debanu Accelero Biostructures > On Jan 31, 2017, at 8:05 AM, sanjeev kumar <sanjeev....@gmail.com> wrote: > > Dear all, > > I am trying to stabilize a protein-protein complex. Our SPR study indicates > it is having micro molar dissociation constant. I tried to purify both the > molecule in complex form with size exclusion chromatography (mixed both the > protein in equal molar ratio and incubated at 4 degree for 1 hour), I didnt > observed formation of complex as both the molecule eluted at their respected > elution volume. > Please suggest me to get a better way to achieve the complex and if anyone > gives idea about what is the good cross-linker I can use. > Suggestions are highly appreciated. > > Thanks > > best > sanjeev kumar, PhD > Purdue University > West Lafayette > Indiana > >
