Multiwell filtration system results in rapid, high-throughput rubber transferase microassay
2000
Mau, C.J.D. | Scott, D.J. | Cornish, K.
The amount of rubber synthesised by washed rubber particles (WRP) has been measured by the incorporation of radiolabelled isopentenyl diphosphate in enzymatic reactions assembled in a MultiScreen 96-well plate with the wells sealed underneath with a membrane which prevented leakage but allowed rapid harvesting. After stopping the reactions, the WRP were trapped on the underlying membrane by applying vacuum and, after appropriate processing, the radioactive rubber on the filters was determined by liquid scintillation counting. The processing time for 96 samples was less than 30 min (a 20-fold reduction compared with our previous published protocol) and, compared with another multiwell rubber transferase assay, the present method yielded a 4-fold saving in time and eliminated the requirement for carrier rubber. The described assay can measure accurately the activity present in 0.07 mg of WRP and can he performed in a volume of 5 microliter, which is smaller than any other assay reported. This assay can also be used to measure rubber transferase activity in divergent species, such as Hevea brasiliensis, Parthenium argentatam and Ficus elastica.
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