I meant "where the drops have the concentration of ammonium acetate needed".
James On Nov 9, 2012, at 10:06 AM, James Stroud wrote: > 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 >> >
