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Wei,

I am not sure if I can solve your problem, but I can try to give insight.

I am also working with a similar protein-DNA complex situation.  I normally purify the protein by itself, and maintain the salt at 250 mM or greater.  Addition of 1.5x DNA stabilizes this complex and I am able to dialyze the sample into 50 mM salt with no precipitation issues.

In addition, I always check every piece of DNA using fluorescence anisotropy to make sure it actually binds.

To comment on your points:

1) this is a common issue I think with most DNA binding proteins.  Addition of DNA *usually* stabilizes the protein and allows for lower salt.

2) are you certain that your protein can bind DNA at high salt? usually high salt decreases the Kd since you are disrupting the electrostatic interactions made between the dna binding domain of your protein and the dna itself.  Also, I would try incubating on ice instead of RT. In addition, what are the differences in the DNA buffer and protein buffer? Are there large pH differences? Perhaps your protein doesn't like basic solutions, which most DNA is normally annealed/resuspended in.

3) Since you are seeing a large DNA elution peak on your sizing column, this almost suggests to me that your DNA is not binding.  This may seem like a dumb thing, but I would check and make sure that your protein can actually bind the DNA sequence you are trying, using something like FA/FP and a labelled oligo.

4) Again, this makes me think it isn't really binding the DNA, since your protein is crashing out.  I think you could check this if you do a high-speed spin to pellet the protein precipitant and check it to see if the DNA is crashing out with it? Or check the supernatant to calculate the DNA concentration.

These are just some suggestions I can think of off the top of my head,

Bret

On Tue, May 22, 2012 at 10:03 AM, Wei Huang <[log in to unmask]> wrote:
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