It is not very surprising that the affinity gets higher with lower salt, right? Why dont you measure it under _physiological_ salt concentration? (or i assume maybe you did?) and of course its not as high affinity due to screening (but physiological conditions) of the electrostatic interactions.
If the complex doesnt stay together in ca. 150 mM salt (e.g in TBS) in gel filtration (which you dont say if you tried) then why not just try mixing it in the drop and screen, this is what you would do if you cant purify the complex. and try different rations. There are texts on preparation of DNA complexes in case you dont have advice for it. Tommi On Nov 9, 2012, at 6:50 PM, Tim Gruene wrote: > -----BEGIN PGP SIGNED MESSAGE----- > Hash: SHA1 > > Dear Wei Huang, > > if you are lucky you can form the complex as crystal in the drop (I > assume you want to crystallise the protein-DNA complex): > > set up drops at high salt concentration (as low as possible to keep > the protein in solution at reasonable concentration) in the presence > of DNA. > > Prepare the buffer the same way as the protein buffer, but with > reduced salt concentration (btw, you can also try divalent ions, eg. > MgCl2). > This way water evaporates into the drop, diluting the salt concentration. > > - From your discription the solubility of the protein drops faster than > its concentration, hence it should precipitate. And, as I said, if you > are lucky, it does so as a crystal in complex with the DNA. > > Best, > Tim > > On 11/09/2012 05:14 PM, 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, >> > > - -- > - -- > Dr Tim Gruene > Institut fuer anorganische Chemie > Tammannstr. 4 > D-37077 Goettingen > > GPG Key ID = A46BEE1A > > -----BEGIN PGP SIGNATURE----- > Version: GnuPG v1.4.12 (GNU/Linux) > Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ > > iD8DBQFQnTRFUxlJ7aRr7hoRAvscAJ9Bm/3ix2ln69wZz74LWwaPMdBhFQCfS9C6 > nvrZMfMmeekAauJ26jy6jbE= > =nbV+ > -----END PGP SIGNATURE----- Tommi Kajander, Ph.D., Docent Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi http://www.biocenter.helsinki.fi/bi/kajander/
