Thanks all of you for your answers. As you guessed I was doing coomassie staining from single crystal (0.10-0.13-0.05).Fortunately I had lots of not so great looking single crystals from similar drops and I took about 10-12 of them.Then I could see a faint band where I was expecting!!!!! Thanks again.
Ivan On Tue, Nov 2, 2010 at 9:15 AM, Kenneth Verstraete < [email protected]> wrote: > Hi Ivan, > > there are several tests (e.g. Izit dye, crush test) you can do discern > protein from salt crystals but what was always very informative to me (and > certainly in the case of complexes) is a silver-stained SDS-PAGE gel of the > crystals using the following protocol: > > - select a drop which contains some substantial crystalline material. The > crystals can be many and small (crystal shower) or few and large. > - prepare a PCR-tube with eg. 50 microliter stabilizing buffer (mother > liquor containing a 10% higher concentration of precipitant) > - transfer all the crystalline material from the drop into the PCR-tube > using a pipet (use stabilizing buffer from the PCR tube to collect all > crystals) > - centrifuge the PCR-tube at low speed for 30-60 sec and observe the > crystals under the microscope. They should be at the bottom of the PCR-tube. > - Remove as much as supernatant as you can (make sure not to remove your > crystals), add stabilizing buffer to wash the crystals, and centrifuge again > - repeat this washing protocol a few times > - after the final washing step, add Laemli-buffer to the crystals and use > this sample to load the SDS-PAGE gel > - include a positive (eg. solubilize another drop directly in > Laemli-buffer) and a negative (final washing buffer) control > - use silver staining to visualize the protein > > This always work for me. If you don't see a band a this point I would be > worried that it is salt. You could then choose to do a Western blot in stead > of silver staining to increase the sensitivity. Make your to include control > samples then. > > Kind regards, > > Kenneth Verstraete > L-PROBE > Ghent University > Belgium > > > Citeren "xaravich ivan" <[email protected]>: > > > Hi everyone, >> I have grown some crystals after micro-seeding starting from thin-small >> needles from needle-clusters. These crystals are larger in size than the >> needles but are comparable to the shape and don't look like salt crystals. >> But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a >> home source,handy and would like to send these to the synchrotron. >> >> Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, >> the amount of protein is < 1uG? >> Has anyone experienced such a thing (no band in gel, but crystal >> diffracts)? >> >> It would be nice if I get observations/suggestions. >> >> ivan >> >> > > >
