You could also try co-expressing them, pull down the complex by his tag. Also you can try equilibrating the SEC column in a low concetration of one of the proteins, if you can express enough. It is a bit surprising that you saw no peak for the complex though. Since you have SPR data, what are kon and koff—fast I assume?
JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ursula Schulze-Gahmen Sent: Tuesday, January 31, 2017 11:30 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] For stabilizing protein-protein complex We have had good luck with creating fusion proteins of the 2 proteins in question (http://www.nature.com/articles/ncomms14076). If you don't know how they interact, you would need to try different linker length and different order of the 2 proteins in the fusion protein. It would also be helpful to have a way to assay for the functional complex. Ursula Schulze-Gahmen On Tue, Jan 31, 2017 at 8:05 AM, sanjeev kumar <sanjeev....@gmail.com<mailto: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 -- Ursula Schulze-Gahmen, Ph.D. Project Scientist UC Berkeley, QB3 360 Stanley Hall #3220 Berkeley, CA 94720-3220 (510) 643 9491
