Yes, oil is great, but you have to be careful to choose an oil in which
the alcohol is not soluble, or the oil will suck it out of your drop,
(just like air). This is particularly annoying with detergents, which
are almost all soluble in oil. I've always thought that maybe some
synthetic motor oils (which your auto mechanic will tell you are
immiscible with petroleum-based oils) might be a good thing to try with
membrane proteins.
It is a common trick, however, to pre-saturate the oil by vortexing it
with an excess of the reservoir solution before applying it to the
drop. Obviously, however, this trick can get expensive when working
with detergents...
-James Holton
MAD Scientist
Nathaniel Clark wrote:
I have wondered if placing a layer of oil over the drop would help
solve the problem of the crystals moving around. Haven't tried it,
but don't people harvest from a microbatch tray by dragging the loop
and crystal through oil?
Nat
On Fri, Apr 9, 2010 at 11:21 AM, James Holton <jmhol...@lbl.gov> wrote:
Yes, isopropanol is a cryoprotectant, and a relatively good one. So are the
other alcohols. It was even popular in the "olden days" when we would
typically set up drops that were 5-10 microliters in volume (each!). These
take a while (minutes) to evaporate, giving you enough working time to mount
the crystal before the alcohol concentration changed "too much". Modern
nanoliter-scale drops have largely made alcohol additives impractical, which
is a shame.
A potentially general way to deal with evaporating drops is to bathe the
work area in a stream of air or nitrogen that has been pre-saturated with
the reservoir solution. That is, run the gas line in and out of a jar of
say about 50-100 mL of replicated reservoir solution (bubbling the gas
through the solution in the jar) and then route the end of the hose to under
your dissecting microscope and point it at your crystallization well just
before you crack it open. This should give you a nice, long working time,
and similar devices have already been reported in the literature:
http://dx.doi.org/10.1107/S0021889801020702
That, or you can try to just work really quickly!
-James Holton
MAD Scientist
Chris Meier wrote:
Dear all,
I have a protein which crystallizes in 25% isopropanol, at pH4.5.
Does anyone have experience freezing crystals grown in such a condition?
What cryoprotectants should I try? Can isopropanol itself act as a
cryoprotectant? Any suggestions on how to deal with isopropanol evaporation
during mounting?
Many thanks and best wishes,
Chris
