Several people have asked about the concentrations we use for various
dyes. In the case of fluorescein, we started with 45mg/ml as a stock
solution. This is quite arbitrary and we often found that our initial
stock solutions needed to be diluted 1:10 or 1:100 before use. It is
more art than science. We also did most of our work actually growing
crystals in the dye rather than using as we would IZIT. So the short
answer is: you'll have to experiment.
You want to inject just enough dye into the crystallization drop to
give a mild coloration. It is probably wise to dilute your stock with
well solution, then inject the smallest volume possible so as not to
dissolve your crystal. Then, over time the drop becomes clear as the
protein crystal absorbs and concentrates all the dye. If you put too
much dye in the drop, the drop is not as clear and it is harder to
distinguish crystals from colored precipitate. We have also seen
crystals of dye form under higher concentrations.
I am not aware of any salt crystals that are known to take up dye. It
is presumed that the dye is absorbed by the protein crystals because
of solvent channels (of which salts have none). We observed even very
low solvent content crystals to take up dye. A negative result,
however, does not rule out protein. We did see a case in which dye
prefers to remain in solution rather than in crystal. I believe this
was high PEG conc.
Under these typical IZIT-like staining conditions, we have not
observed any anomalous signal due to heavy atoms. This is presumably
because the concentration of dye in crystal is too low. At least,
that argues against any dramatic changes in crystal quality as a
result of dye.
Richard
On Jun 16, 2008, at 11:18 AM, Jim Pflugrath wrote:
Whatever dye(s) you use, be sure to run some positive and negative
controls to see how the dye really works.
Jim
On Sat, 14 Jun 2008, Mark Del Campo wrote:
Before I place an order for some Izit, are there some other dyes I
can use to check if I've got a protein crystal?
Thanks,
Mark
