Dear CCP4ers, I am working on purifying a protein-DNA complex for structural and biochemical studies. So far, I can readily make protein > 95% pure in high salt buffer. However, I have some problems in assembling the protein-DNA complex.
1) My protein precipitates at low salt buffer. I think this is the source of all my problems. 2) So I tried DNA binding at high salt buffer. Unfortunately, my protein precipitates after adding DNA (1.2:1 ratio, and DNA duplex was annealed by heat and slowly cool) and incubating at RT for 10 mins. 3) Then I found some posts here and tried DNA binding at very low concentration (< 0.2 mg/mL protein) at high salt buffer, which seems to alleviate the precipitation. And I concentrated protein and DNA mixture, and loaded it on to FPLC to further purify protein-DNA complex. However, I didn't observe the shift of my protein peak upon DNA binding (my protein is a 76 kDa dimer, and DNA duplex is 11 kDa) and I can see a high DNA peak at the position of 17kDA protein standard peak. Therefore, my protein doesn't bind DNA at high salt as expected. 4) The next thing I tried is having DNA binding at low protein concentration while dialyzing in high salt buffer. And slowly titrated in low salt buffer (1mL/min) to a final concentration of 100 mM NaCl. However, this doesn't solve the problem of protein precipitation and I still lose majority of my protein resulting in the purity is < 60%. BTW, the 5% impurity band looks like to be HSP (nearby 75kDa protein marker on an SDS-PAGE). But it seems that the HSP doesn't bind to my protein since my protein still forms dimer. I think it just co-elute with my protein because my last step purification--size exclusive column cannot separate them. If you have come across similar problems and found ways to solve the problems, could you share your experience with me? Thank you very much! I really appreciate your help. Best, -- Alex Huang Research Associate Institute for Cancer Research Xi'an Jiaotong University Xi'an, Shaanxi 710049 China
