On Tue, Nov 2, 2010 at 9:15 AM, Kenneth Verstraete <
[log in to unmask]> 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