Dear,
So what is the problem? to be honest with you, I have hardly heard that someone use hanging drop vapour diffusion to glow 2D crystal because usually you ll end up have 3D crystal or severe stacking problem.
I guess one of your major problem might be stacking, If this is the case, you should try to use lipid monolayer technique. You can check original streptavidin paper by Darst SA and Kornberg RD. In this technique, it relies mostly on how homogeneous your protein is (which I believe you already have it) and the sufficiently good binding of the protein on the surface of the lipid monolayer which you have to figure out (either using electrostatic interaction or employing different tags).
One good thing about his technique is that if you want to solve structure of this protein using electron crystallography, then you ll never have problem with figure out how to make it to stick to your grid which is one of the major problem many 2D people have encountered including me :P.
Hope that helps,
Puey
On Mon, Jan 12, 2009 at 10:24 AM,
<[log in to unmask]> wrote:
Hi,
Thanks for the suggestion. Here is more info: It is a soluble protein, a monomer has 3alpha and 3 beta subunits, and exists in solution as a dimer. The technique is hanging drop, vapor diffusion. And the mother liquor is ~ 0.1 mM cacodylate pH 6.2ish, MgCl2 and PEG 20k.
=v=
-----Original Message-----
From: Puey Ounjai <
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To:
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Sent: Fri, 9 Jan 2009 3:56 pm
Subject: Re: [ccp4bb] 2D
Hello,
The information you provided to us here is not enough. there are so many
parameters and
so many means to optimize 2D crystal. All of which depends on what kind of
protein you
are working on (membrane associated or soluble) and what kind of technique that
you use
to grow your crystal (lipid monolayer, dialysis or else).
I guess unless you give us more info about your crystal otherwise it will be
quite difficult
for us to help you optimize your condition.
Sincerely,
Puey