This sounds like a job for ammonium acetate. Use it as your salt. Purify your complex in it and then set up drops where they wells have the amount of ammonium acetate needed to keep your protein stable and the wells have none, or a range of concentrations. The ammonium acetate will equilibrate by vapor diffusion, lowering the concentration in the drop and causing your complex to come out of solution.
James On Nov 9, 2012, at 9:14 AM, Wei Huang wrote: > Dear CCP4BBers, > > I have a problem in purifying protein-DNA complex for a protein that I am > interested in. > > The purification of protein only has been optimized and I've get enough yield > for what I need (10 mg/2.4 L growth). And I've measured DNA binding using > Fluorescence Anisotropy. The results show that my protein has the tightest > binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES > (pH 7.5). > > However, I came across several problems when I assemble protein-DNA complex > in large scale. > > First, my protein is unstable at low salt condition. When I dialyzes my > protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, > the protein precipitates. What I don't quite understand is that the DNA > binding assay performed at low salt condition doesn't seem to be affected by > this instability of protein. I guess it may be due to the assay was performed > at very diluted protein concentration (in nM). > > Second, I can not purify protein-DNA complex at high salt condition with gel > filtration column. Because of the first problem, I tried to assemble the > complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the > elution profile shows no binding of DNA to my protein (no increase in the > observation of protein peak and a large peak around expected position for > DNA). This may be due to weaker binding at high salt as my DNA binding assay > shows that the Kd under this buffer condition is ~1100 nM. > > Third, a lot of protein is lost during dialysis of protein-DNA complex into > low salt condition. I tried add DNA directly into protein in high salt > buffer, then dialyze very slowly against low salt buffer. However, I still > lost quite a lot of protein due to precipitation. I was able to load some > sample onto the gel filtration column with low salt running buffer. And I saw > the shift of protein peak in the elution profile, also protein concentration > measured by Bradford assay shows that the protein concentration is much less > than that expected from uv trace, suggesting the contribution to the > absorbance from DNA. But the yield is very low, less than 0.2 mg of protein > is left and the complex seems to be unhappy when I concentrate it. So I can > not get protein sample concentrated enough for my study. > > My previous experience with another DNA binding protein is much better. I > purified it in high salt, dialyzed into low salt to binding DNA and finally > purify with gel filtration column. However, the one I am currently working on > seems to be very picky. If you have any suggestion regarding to my problems, > I will be thankful. > > Best regards, > > -- > Wei Huang, PhD > Postdoctoral Associate > Center for Proteomics and Bioinformatics > Case Western Reserve University > Cleveland, OH 44106 >
