Multiwell filtration system results in rapid, high-throughput rubber transferase microassay

The amount of rubber synthesised by washed rubber particles (WRP) has been measured by the incorporation of radiolabelled isopentenyl diphosphate in e nzymatic reactions assembled in a MultiScreen(R) 96-well plate with the wel ls sealed underneath with a membrane which prevented leakage but allowed ra pid harvesting. After stopping the reactions, the WRP were trapped on the u nderlying membrane by applying vacuum and, after appropriate processing, th e radioactive rubber on the filters was determined by liquid scintillation counting. The processing time for 96 samples was less than 30 min (a 20-fol d reduction compared with our previous published protocol) and, compared wi th another multiwell rubber transferase assay, the present method yielded a 4-fold saving in time and eliminated the requirement for carrier rubber. T he described assay can measure accurately the activity present in 0.07 mg o f WRP and can he performed in a volume of 5 muL, which is smaller than any other assay reported. This assay can also be used to measure rubber transfe rase activity in divergent species, such as Hevea brasiliensis, Parthenium argentatam and Ficus elastica.