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of this method is that it cannot be applied at all in cases where volatile
organic molecules (which are soluble in the oils, e.g. dioxane, phenol,
thymol) are required in the crystallization medium, nor with organic pre-
cipitants or additives which interact with the oil.
Harvesting and mounting of crystals
Harvesting crystals from under oil is somewhat more difficult than har-
vesting from a coverslip or a Cryschem plate; hence, a detailed protocol
for harvesting and mounting crystals from the oil has been reported by
Shaw, Stewart (1995). A common problem is the sticking of crystals to
their supporting surface. The standard procedure is to use microtools to
gently release the crystals. Growing crystals in suspended drops
between two oils (as described above and in Fig. 4c) would solve the
problem of sticking but may cause other difficulties. An aid to harvest-
ing crystals which seems to have been overlooked (at least in harvesting
from microbatch), was reported back in 1984 by Ray, Puvathingal
(1984), who found that a layer (2 mm thick) of high-vacuum silicone
grease (which has a gel-like texture) provided an excellent support on
which to grow protein crystals and facilitated subsequent harvesting.
The authors also used the silicone grease to orient seed crystals of phos-
phoglucomutase.
Crystallization of membrane proteins under oil
Crystallization of membrane proteins under oil has not been widely
attempted due to doubts about the suitability of an oil-based method for
crystallizing lipophilic compounds. Surprisingly, a considerable number
of proteins have been successfully crystallized under oil (e.g. Snijder,
2003; Stock
et al
., 1999), some which could not be produced by other
crystallization methods. It is possible that the oil is essential in driving the
process, by slowly absorbing the detergent from the aqueous drop, thereby
encouraging the protein to gradually come out of solution and crystallize
(Hankamer, personal communication).
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