Author: Stewart, Meredith E.; Roy, Polly
Title: Structure-based identification of functional residues in the nucleoside-2'-O-methylase domain of Bluetongue virus VP4 capping enzyme Document date: 2015_2_24
ID: vzel6r43_28
Snippet: To examine the formation of cap structures by recombinant proteins in vitro, capping assay was performed using the standard methyltransferase assay as described [4] with the following modifications: 5 lg of recombinant protein, 0.5 lCi/ll a 32 P-GTP and 1 lg RNA. After incubation for 2 h at 37°C, RNA was purified and digested with NucleaseP1 (Sigma) to release the cap structure. Cap structures were resolved by thin layer chromatography (TLC) usi.....
Document: To examine the formation of cap structures by recombinant proteins in vitro, capping assay was performed using the standard methyltransferase assay as described [4] with the following modifications: 5 lg of recombinant protein, 0.5 lCi/ll a 32 P-GTP and 1 lg RNA. After incubation for 2 h at 37°C, RNA was purified and digested with NucleaseP1 (Sigma) to release the cap structure. Cap structures were resolved by thin layer chromatography (TLC) using PEI-cellulose plates (Millipore). Reactions were performed in triplicate and ImageJ was used to quantify the intensity of the cap structures generated [30] . Data was analysed for significance using student t-test.
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