I do not know about the affinity required for gel filtration, but I have had success crystallizing a number of protease-inhibitor complexes just by incubating and setting up drops with equimolar amounts of the two components. It is probably to our advantage that we have functional inhibition assays that allow us to be very precise in achieving the equimolar ratio. This has worked for a lot of complexes with Kd in the low nanomolar to low picomolar range, but we recently also had success with a complex where Kd was 14 micromolar.
Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Ngo Duc Tri Sent: Wednesday, September 19, 2007 6:20 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Question regards to binding affinity of the protein complex? Dear CCP4 Users, I'd like to solve the structure of the protein-protein complex. I intend to purify and incubate the complex then run gel-filtration before setting crystal. I'd like to know your experience about the Kd value of the interaction in order to get the complex after running the gel-filtration. In other words, what is the lowest Kd that we can purify the protein complex using gel-filtration? And if the binding affinity is too low to purify the complex, is it possible to get the crystal of the complex if I just incubate and set crystal of this mixture? Thank you very much for all of your advice! My best regards, TriNgo Sungkyunkwan University
