Dear TriNgo, The Kd is only 1/2 of the picture. You also should contemplate the consequences of the on and off rate for the complex formation. If the off rate is in the range of minutes then you can have successful purification over size exclusion even though the Kd may be high micromolar.
Other considerations include the ratio between the plate volume of the sizing column (a theoretical number but useful nevertheless) and the volume of the loaded complex, as well as the concentration of the latter and the rate of diffusion in the column. This is a sticky question, and ultimately the proof is in the pudding - do the experiment on a small scale and check whether the complex comes out 1:1 or you get individual components as well as some ill-defined ratio in the higher m.w. range. As for the second question - yes, many people have co-crystallized complex components by simply mixing them in the (approximately) right ratio. Keep in mind that this ratio isn't necessarily 1:1 - some people only had success with great excess of one component over the other, whereas others found that 1:1 worked best. Therefore in my opinion your best chance to find the right solution is to try a few ratios in a simple screen (say, one 96-well screen over 6 or 9 ratios) to see how things develop from there. Good luck, Artem > 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 >
