Dear Chen, how sure are you that your crystals contain the protein of interest? Repeatedly washing harversted crystals in crystal stabilization solution followed by SDS-PAGE coupled to appropriate staining protocols (e.g. Coomassie, Silver staining) or western blotting can give a pretty conclusive answer. In our group, SDS-PAGE followed by Silver-staining is done routinely and in most cases leads to conclusive results. Here is a summary of the protocol:
-------- - select a drop containing substantial crystalline material. The crystals can be many and small (crystal shower) or few and large. - prepare a PCR-tube with crystal stabilization buffer (e.g. 50 uL of motherliquor containing a 10% higher concentration of precipitant). - transfer all the crystalline material from the drop into the PCR-tube using a pipet (You can use stabilization buffer from the PCR tube to collect all crystals). One can also use a cryo-loop to harvest the crystals if they are large enough to allow efficient harvesting. - centrifuge the PCR-tube at low speed for 30-60 sec and observe the crystals under the microscope to make sure they are at the bottom of the PCR-tube. - remove as much of the supernatant as you can using a pipet making sure not to remove the crystals. Then add crystal stabilization buffer to wash the crystals, and centrifuge again. - repeat this washing procedure a few times (typicaly 3-4 times). - after the final washing step, centrifugation and removal of the supernatant, add Laemmli-buffer to the crystals and use this sample for loading onto the SDS-PAGE gel. - include a positive control (e.g. solubilize another drop directly in Laemmli-buffer) and a negative control (final washing buffer). Including a pre-crystallization sample of your protein as a control is also recommended, to control for the integrity of the protein under crystallization conditions. - use silver staining to visualize the protein. ------- best regards Savvas On 31 Mar 2014, at 23:48, Chen Zhao <[email protected]> wrote: > BTW, I forgot to mention that phenix.autosol also gave similar result. > > > On Mon, Mar 31, 2014 at 5:46 PM, Chen Zhao <[email protected]> wrote: > Dear all, > > I am now trying to phase a structure in C2 using anomalous scattering at 5-6 > A. It is hard to improve the derivative resolution at the moment. Shelxd is > able to locate 6 sites with a distinct CC and FOM. After density modification > in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and > 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and > MAD are similar, and the solvent boundary is quite clear. However, the > problem is that the electron density blob passes through the 2-fold rotation > axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small > for the molecule. I am afraid that the space group assignment is wrong, but I > am a beginner so I nearly have no clue. I did reprocess the data in P1 and > looked at the self-rotation function with a radius at 200 A. From the list it > seems that there is only one 2-fold rotation axis. I am quite confused. Could > anybody give me some hint of this problem? > > Thanks a lot in advance! > > Sincerely, > Chen >
