Dear CCP4ers,<div><br></div><div>I am working on purifying a protein-DNA complex for structural and biochemical studies. So far, I can readily make protein &gt; 95% pure in high salt buffer. However, I have some problems in assembling the protein-DNA complex.</div>
<div><br></div><div>1) My protein precipitates at low salt buffer. I think this is the source of all my problems.</div><div><br></div><div>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.</div>
<div><br></div><div>3) Then I found some posts here and tried DNA binding at very low concentration (&lt; 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&#39;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&#39;t bind DNA at high salt as expected.</div>
<div><br></div><div>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&#39;t solve the problem of protein precipitation and I still lose majority of my protein resulting in the purity is &lt; 60%.</div>
<div><br></div><div>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&#39;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.</div>
<div><br></div><div>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.</div><div><br></div><div>Best,</div>
<div>-- <br><font face="&#39;courier new&#39;, monospace">Alex Huang</font><div><font face="&#39;courier new&#39;, monospace">Research Associate</font></div><div><font face="courier new, monospace">Institute for Cancer Research</font></div>
<div><font face="courier new, monospace">Xi&#39;an Jiaotong University</font></div><div><font face="courier new, monospace">Xi&#39;an, Shaanxi 710049</font></div><div><font face="courier new, monospace">China</font></div>
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