I would try it both ways and see what you get. Also do controls of buffer into each protein
For extra info could also try with SPR. Always best to do these things using multiple complimentary methods Best wishes Clare From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Bernhard Rupp Sent: 03 October 2019 16:06 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] ITC question -dimer vs monomer Hi Fellows, please let me ask the respective experts an ITC question: I have 2 proteins, stable and dialyzed in identical buffer. A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer will form. Intuitively, it should make a difference whether I titrate the dimer with the monomer or vice versa. In the first case, a momomer would initially meet a lot of free dimers, and I would expect that randomly, a AB2 complex is more likely to form than a A2B2 (let's disregard any more complex colligative/cooperative effects). If I drip the dimer into the monomer pool, it is quite likely that the B dimer meets 2 free As, and I get right away a higher population of A2B2s. Maybe at dilutions of ITC and with sufficient equilibration that is not an issue at all (again, absent any cooperative effects that might alter the first Kd vs. the second, despite the sites on the dimer are at least initially equivalent). Can someone guide me towards literature about this or perhaps share some first-hand experience? Many thanks, BR ------------------------------------------------------ Bernhard Rupp http://www.hofkristallamt.org/ b...@hofkristallamt.org<mailto:b...@hofkristallamt.org> +1 925 209 7429 +43 676 571 0536 ------------------------------------------------------ Many plausible ideas vanish at the presence of thought ------------------------------------------------------ ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
