If your SPR data is correct, you'll need to use a micro molar protein concentration (or higher due to dilution in the column) in your analytical size exclusion chromatography assay. What concentration did you use? Also, ideally the buffers in both assays should match. And maybe your SPR data is the source of confusion (it is not uncommon). What is the shape of your sensorgrams? Do you measure the dissociation for how long, or does it happens abruptly? Napo
----- Mensagem original ----- > De: "sanjeev kumar" <[email protected]> > Para: [email protected] > Enviadas: Terça-feira, 31 de Janeiro de 2017 14:05:32 > Assunto: [ccp4bb] For stabilizing protein-protein complex > 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
