Go to Hampton Research website to check materials for protein crystallization.
Normally, you need to get pure protein first;
then do crystal screening to see if you could get protein crystal;
then optimize the crystallized conditions to get good single crystal;
then collect X-Ray diffraction data at synchrotron beamline at Beijing or other synchrotron facilities all over the world, or lab-based X-ray equipment;
then solve the protein structure by softwares(if you really want to do the thing, be familiar with softwares, such as CCP4, CNS, SHELX, COOT, solve/resolve and others, from now on. Some softwares are free to academia so you can download for their websites, but some not free, if you boss has enough money, just ask to buy, but I think many bosses in China are miser).
then deposit you structure to PDB (protein data bank), and publish your paper if you get the structure. If you can do the relative functions, that is better. Structure plus function is the best. A very good paper will you get.
Anyway, the experiments are routine. You have to spend more time on softwares if you want to be an expert.
Besides, I think you need to read some textbooks about protein crystallography. Basic knowledge is necessary.作者: wmp1234 时间: 2013-11-21 16:47
Many companies have commercialized screening kits, such as Hampton Research, Emeralds, Qiagen. If you do not want to buy the Hampton Research screening kits, you could prepare the kits according to the formula. But the total cost for buying reagents would be more than that of buying the screening kit. The best way is to co-operate with the labs which do protein crystallography because of your lab's condition. Some labs in Beida, Qinghua, and Institue of Biophysics do that job.
If you have other questions later, just free to ask.作者: bluelake 时间: 2013-11-21 16:48
What tag did you use for your protein? Histag, GST, MBP or others? You have to check the purity with SDS-PAGE after every purification step, such as Ni-NTA column, Ion-exchange, and size column. As xsvulture said, the purer, the better.
There is one thing you may be confused. The 95% purity on SDS-PAGE does not just mean enough purity. Because the purity on SDS-PAGE is based on denatured form. The 95% purity should include the homogeneity of three-dimensional shape and the charge distribution of protein.
After you get good purity (>95%) on SDS-PAGE, you maybe need to run a size-column to check the apparent molecular weight of your protein to see if it is dimer, tetra-mer or aggregate(for example, big M.W. more than one million) (In aggregate case, you won't get any crystal even though you can get only one band on SDS-PAGE. I met the case before). This way is crude.
There are other ways to check the homogeneity of protein in certain buffers, such as Dynamic Light Scattering and thermofluor method. You can check the references later if you really need to do these experiments.
Just do crystallization as the references' method first. If no crystal, do crystal screening.
How much is the sequence identity of your RNaseA to the crystallized one? I think that there should be structure of the latter in PDB and maybe you could use Molecular Replacement to solve the structure of your RNaseA if you could get crystal later.
