Many protein have been seen as a complex in coIPs but the complex could not be obtained with the purified components. In the coIP you never know how many other different proteins were present in the complex bc they usually use western blotting to test for the presence of a particular set of individual proteins. was the coIP analysed by mass spec? I would consider that you might miss one or more proteins that act as bridging components to stabilise your complex. If you cannot demonstrate that the complex forms in solution: pulldown, SEC, AUC, ITC, …. how do you convince the reader later that you complex in the crystal is physiological relevant?
Best, Sebastian > On 15 Jan 2016, at 07:54, George Kontopidis <[email protected]> wrote: > > Dear Chen, > > The two proteins try to be crystallized, are exactly the same construct with > the two proteins have been tested in co-IP? > A his tag may affects binding. > > The two proteins have been tested in co-IP were expressed in E.coli? > Post translation modification for sure effect function and thus binding > > The PUll-down assay or gel fitration was taken place at the same or similar > pH and salt concentrations with those of co-IP ? > Usually in gel filtration medium or relative high salt concentration is used > > George > > -----Original Message----- > From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of <Xia > Chen> > Sent: Thursday, January 14, 2016 1:34 PM > To: [email protected] > Subject: [ccp4bb] how to enhance the complex interaction(weak) > > Dear every one: > nearly, I have try to crystalize two protein complex. Although we get > know from the cooperator that two protein have interaction just for > Co-immunoprecipitation (IP). when I express them in E.coli. regardless of > PUll-down assay or gel fitration, we can't dectect the interaction. I want > to find some ways to enhance their interaction so that I can crystalize the > complex. > > > > Chen Xia, Ph.D > > School of Life Sciences > Tianjin University > 92 Weijin Road, Nankai District, Tianjin 300072, P.R.China
