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Hmmm is it really (physiological-like) "binding", or your protein A is aggregating/precipitating on the "partner"? Do you have a good negative control (a similar protein to which protein A should not bind)? Also, as a general rule, be careful about your detection method for the pull-down, don't get too sensitive or you'll start seeing hair on eggs -- maybe that's not the case, since you say "strong interaction". Carlos Em 22 oct. 2013, ąs 04:39, Clement Angkawidjaja <[log in to unmask]> escreveu: > Dear Dee, > > Some proteins with chaperone-like activity (perhaps your B?) can only bind to partially folded proteins. > Probably A folds to a molten globule structure after 1-2 days. You can check by spectroscopic techniques (ANS or Trp fluorescence, CD). > Hope that helps. > > Cheers, > Clement > > On 10/22/13 11:10 AM, Xiaodi Yu wrote: >> Dear All: >> >> I have a general question about protein- protein interactions. I have two proteins, A and B. A is a disordered protein while B is a well folded protein. The binding between A and B has been approved by GST-pull down assay previously. The strange thing is I cannot get them bind if protein A were just freshly prepared. However, if I kept these two proteins separately for one or two days at 4 degree and then did the GST-pull down assay again, I can observe very strong interaction between A and B. >> >> Protein A doesn't contain any cys residue. I have already test certain chemicals which might affect the interactions, for example, DTT and EDTA. These chemicals seems to have no effect on the binding. >> >> Although A is a disordered protein, does it need such long time to find its proper conformation? >> >> Do any people have similar experience? Any suggestions are greatly appreciated. >> >> Thanks, >> >> Dee >