Assay Method Information

Assay Name:  RSV Polymerase Assay (RSVpol)
Description:  Standard RSV polymerase assays were conducted in the presence of 3 μL extract of RSV-infected cells in a reaction buffer containing 50 mM tris-acetate pH 8, 120 mM K-acetate, 4.5 mM MgCl2, 5% glycerol, 2 mM EDTA, 50 μg/ml BSA, and 3 mM DTT. Varying concentration of NTPs were used to initiate RNA synthesis for 120 minutes at 30 degrees, and radioactive 33P GTP (15 μCi) was used as tracer. The reaction was stopped by adding 50 mM EDTA, and RNA samples were purified through G-50 size exclusion spin columns and phenol-chloroform extraction. The radio-labeled RNA products were resolved by electrophoresis on a 6% polyacrylamide TBE gel, and visualized and quantitated after being exposed on a phosphorImager screen. Polymerase inhibition experiments (IC50s) were conducted the same way in the presence of increasing concentration of NTP analogs.
Affinity data for this assay
 

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