Dear Savvas, Thank you for your reply and your nice protocol. I am also worried of this problem so I have already run my crystals on the gel for several times. The result is so clean that you can hardly draw any other conclusions except that the crystals are made up of the full-length molecule. But possibility remains regarding this concern if my molecule of interest is not in crystalline state but other molecules that cannot be stained are.
Best, Chen On Tue, Apr 1, 2014 at 3:30 AM, Savvas Savvides <[email protected]>wrote: > 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 > > > >
