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components into suspension. The Eppendorf tube was then centrifuged (13.000rpm, 4°C,
10 min, Biofuge pico, Heraeus) and the supernatant was transferred to a clean sterile ice-
cold Eppendorf tube and frozen at -20°C until ready for AFP analysis.
All protein extracts were tested for protein using the BioRad Protein Assay Kit
(BioRad). The assay was based on the Bradford method. It utilizes Coomassie Blue (G-
250) as a dye reagent. The absorbance maximum shifts from 465nm to 565nm in an
acidic solution as the dye binds to protein. Lyophilised Bovine Serum Albumin and Fish
AFP III (10mg/ml) was used as a positive control, while Protein Extraction Buffer was
used as a negative control. The sample (l00µ1) was transferred into a clean test tube to
which 5ml of dilute BioRad dye reagent was added. The sample was vortexed and then
measured at OD595 verses a reagent blank using a spectrophotometer (Cecil CE 2021,
2000 series). The positive controls both showed a deep blue colouration, while both
negative controls showed a brown/red colouration consistent with the cationic, double
protonated state of the dye at the assay pH.
2.10.2 - `SPLAT' analysis
The protein extractions (PE) were taken on ice to Unilever's Colworth laboratory
where they were tested for recrystalisation inhibition activity using the `SPLAT' assay
equipment. The protocol is modified from Knight et al. (1988). PE solutions were
standardised by adding 50 µl of 60% sucrose into solution with 50 µl of PE. These were
made up in 1.5m1 fixed lid Eppendorf tubes. The 30% sucrose/PE solutions were spun in
an MSE Micro Centaur desktop micro-centrifuge for 10 sec (to make sure all liquid was
at bottom of the tube and mixed well). Of the resulting solution, 5µl was placed between
two circular 16mm diameter, numbered coverslips, which were blotted dry to remove any
excess fluid. The coverslips were placed in 2,2,4 - trimethyl pentane pre-cooled to -70°C
(using dry ice) for 2 minutes, to super-cool the samples. A circulating solvent bath was
filled 3/4 full of 2,2,4 - trimethyl pentane and pre-chilled to -6°C using a Haake C water
bath circulator and two Haake Temperature control units (PG40 and F4). The coverslips
were taken directly from the -70°
C solvent baths to the -6°C solvent bath (as quickly as
possible to prevent melting) and held there for one hour to allow for recrystalisation. The
coverslips were then viewed whilst in the bath using an EF L 20/0.30 160/0-2 objective
on a Leitz Dialux 20 EB stage. The observed crystal shape, size and density were
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