Hi Rhys,
you already have a lot of suggestions to try. We all have our own
reciepe for good derivatization, and this is due to the fact that we
don't really understand what is going on. I can't explain why one HA
binds to my protein while the other one doesn't, but I can visualize it
and hence choose the good one. And i've observed this behavior with
several proteins already.
I would suggest that you try which HA is best for you, and use this
specific one for your soaks.
Testing different HA is quite easy and can be done simply by running a
gel. you can try native-PAGE if your protein handles it, or we
developped a technique on SDS-PAGE coupled with a fluorescence dye
(http://www.ncbi.nlm.nih.gov/pubmed/20152903).
Using these techniques, you can distinguish easily which HA binds to
your protein (in the later case, free cysteines) and you will have big
surprises. You can test concentration and derivatization time fitting
your needs, you do this at your bench and have the result within an
hour. It's worth investing the time in biochemistry before going to the
synchrotron and processing lots of data to realize it wasn't the good HA
and it won't bind to your protein...
It won't garantee a non-mobile HA in the crystal, but at least you will
try something that has more chances on working.
Don't hesistate to contact me if you need more practical infos.
Good luck
Vincent
On Jan 16, 2014, at 2:18 AM, RHYS GRINTER wrote:
Hello message board,
My group has some crystals of an interesting protein to take to the synchrotron
in a couple of weeks. We won't be able to prepare and crystallise a SelMet
derivative during that time period, but we have loads of crystals sitting
around. The diffraction isn't great, we see maybe 3.5 at home but might be
enough to get over the line.
It will be a very difficult MR target, so we were thinking of soaking so
crystals with heavy atomic compounds that we have lying around. I was wondering
if people had any suggestions of compounds that people have used successfully
for experimental phasing and maybe concentrations to use and soaking time.
Cheers,
Rhys
--
Vincent Chaptal, PhD
Institut de Biologie et Chimie des Protéines
Drug-resistance modulation and mechanism Laboratory
7 passage du Vercors
69007 LYON
FRANCE
+33 4 37 65 29 01
http://www.ibcp.fr
