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
