Dear all,
I have some questions when soaking heavy atom derivatives,
1. How to prepare HA derivative stock solution? I use 10 mM Bis-tris pH 6.8 to
dissolve my heavy atom derivatives ( Hampton Research Heavy Atom Screen M1 and
Heavy Atom Screen Hg), the concentration is 100 mM. Apparently, some of the
derivatives in Screen M1 and many in the Screen Hg are poorly soluble.
2. I ran a native PAGE to have a pre-screen of HA derivatives soaked with
protein solution. Indeed I saw some shifted lands in my gel. My question is:
For the hard ligands which binds protein ionically, will the protein and the
ligand seperate in the gel? If so, the protein become native again and no
shifted land can be observed?
3. The protein is a homotetramer with one cystine in one 200-aa-chain. I have
no idea whether this cystine is reduce or not (maybe I can run a non-reducing
SDS-PAGE). If I can soak it with Hg, are these four Hg atoms enough for
phasing? I was told that approximatly one Se-Methionine per ~100 aa is enough.
Thank you and waiting for your suggestion!
Lu Zuokun
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卢作焜
南开大学新生物站A202
