Hi John, Another way to screen for mercury derivatives.
Rachelle vincent Chaptal <vincent.chap...@ibcp.fr> wrote: 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
