The Role of Oil in Protein Crystallization - Molecules: Nucleation, Aggregation and Crystallization - page 134

can dry up within 24 hours. Paraffin and silicone oils are miscible,
and it was shown by D'Arcy et al ., (1996) that by mixing different
ratios of these two oils the evaporation from a trial drop can be regulated
so that the ingredients in a trial become more concentrated with time until
the eventual drying of the drop. This modification of the microbatch
method provides a means of simultaneously retaining the benefits of a
microbatch experiment combined with the inherent advantage of the self-
screening process of a diffusion trial. The authors reported that using a
combination of paraffin and silicone oils to cover microbatch trials for
screening experiments resulted in the appearance of crystals within a
shorter space of time compared with trials which were situated under
paraffin oil. These results were confirmed by Baldock et al . (1996). A sim-
ilar effect can be accomplished by using silicone oil alone (D'Arcy et al .,
2004) or by varying the thickness of the oil layer covering the trials
(Chayen, Saridakis, 2002). Obviously, when using the combinations of
oils and/or a thin layer of oil, frequent monitoring of the trials is impera-
tive. Once crystals are observed, more oil must be applied to prevent the
drops from drying up.
It is interesting to note that not only can the type and the quantity of
the oil dictate the outcome of a crystallization experiment, but also the
time of incubation. The effect of time as an additional factor was revealed
when crystallizing -crustacyanin, a protein of the lipocalin family, which

could only be crystallized by the microbatch method under paraffin oil,
yet it took four months to produce diffraction quality crystals (Cianci
et al ., 2002). No crystals were produced if trials were set under a mixture
of paraffin and silicone.
It transpired that in spite of being covered by paraffin oil, some evap-
oration was taking place due to the lengthy time of incubation. In a typi-
cal microbatch experiment, crystallization takes place within a week or
two, and since water and paraffin oil are essentially immiscible, evapora-
tion during this time is negligible. However, given ample time, slow evap-
oration can occur (as there is no absolute immiscibility), which can
proceed until the drop dries out. It is apparent that the

β

-crustacyanin
underwent a very gradual concentration until it reached the certain point

β