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Filtrate was then centrifuged for 1 h at 4 °C and 12,000 rpm; the supernatant collected was used to prepare the crude enzyme by precipitating for 24 h at 4 °C with 4 M ammonium sulphate with continuous stirring.
After precipitating for 30 min at 37°C, the mixtures were centrifuged for 15 min 12,000 g and 4°C.
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Then, the mixed solution was stirred for 5 h and precipitated for 15 h.
The suspending solution was precipitated for 12 h and the upper supernatant was collected and centrifuged.
However, the amount of Sc that can be precipitated for hardening is rather limited because of its limited solubility.
Unhydrolyzed protein substrate was allowed to precipitate for 15 min at 4°C and centrifuged at 6,500 × g for 10 min.
The finite element method was employed in order to calculate stress distributions around the SiGe precipitates for different precipitate diameters using both elasticity and plasticity models.
One may therefore assume that an important and universal evolutionary constraint precipitated for the preservation of circadian input functions in the adult.
The mixture was allowed to precipitate for 15 min at 4°C.
After centrifugation, the resulting supernatant was added to 60% saturated ammonium sulfate solution, and precipitated for 4 h on ice.
Based upon the results of the hydroxyproline assay, the equivalent amount of each tissue digest containing 20 µg of total collagen was precipitated for blotting.
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