Dear Jan I would recommend running the following protocol on your spherulites. Just pretend that they are crystals :) This was posted some time ago on the ccp4bb. best regards Savvas
> On Tue, Nov 2, 2010 at 9:15 AM, Kenneth Verstraete < > kenneth.verstra...@ugent.be> 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 works for me. If you don't see a band at this point I would be > > worried that it is salt. You could then choose to do a Western blot instead > > of silver staining to increase the sensitivity. Make sure to include control > > samples then. > > > > Kind regards, > > > > Kenneth Verstraete > > L-PROBE > > Ghent University > > Belgium On 24 Aug 2011, at 20:05, Jan van Agthoven wrote: > Dear all, > > I recently obtained some spherulites while trying to crystallize my protein. > The spherulites are manually reproducible, but changing pH, protein > concentration, and salt concentration does not result in crystal formation. > Microseeding with crushed spherulites isn't a solution either as it only > yields new spherulites. Next stepp is the use of an optimization kit but I > have a limited amount of material, and I start doubting that these are > protein spherulites, as the spherulites are not particularly soft. The > condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 > forms easily spherulites around that concentration? > > > Thanks,
