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
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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
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
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
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
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
On 11/09/12 15:43, Tommi Kajander wrote:
It is not very surprising that the affinity gets higher with lower
salt, right?
Not at all. I wanted to ask if their assay could distinguish between
specific binding and nonspecific binding, but I decided not to sidetrack
the discussion.
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