Dear Wei,
If i understand your different experiment, you try to obtain your
protein DNA complex at different salt concentration with different
method to reach the final concentration.
I read that you try 150 mM KCl + 150 mM NaCl as high concentration salt,
it result in 300 mM cations and 300 mM Cl. To my mind, and according
your experiment it's too high. But when you try low concentration you
dialized the protein alone in 30 and 100 mM salt concentration. In my
opinion the best way, is to dialize the protein mixed with the DNA.
Because the protein will probably be stablize by DNA. If the protein
meet slowly DNA at the same time the salt concentration decrease, you
minimize the precipitation (it's your third experiment in fact). You
have not precised the final salt concentration with this method. If you
try 30 mM, maybe it's too low, i suggest 100mM Salt. I remember paper
about nucleosome where author explain solution with multiples steps
dializis. It's more gradual and it could help if you want reach very low
salt concentration.
One other idea, is to play with the pH, because of the protein's pI. In
function of the pI you have to try different pH for the complex
preraration. You can optimise the protein DNA interaction through the
protein charge.
HtH
Nicolas
Le 09/11/12 17:14, Wei Huang a écrit :
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
