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The vN�CRNA template was treated with chymotrypsin A (Boehringer Mannheim) at ratios of 4.25:1, 8.5:1 and 17:1 (w/w) (vN�CRNA:chymotrypsin) in the buffer containing 10?mM Tris�CHCl, pH 8.0 at 37?��C for 20?min. Reactions were stopped by adding Laemmli buffer and analyzed immediately by SDS-PAGE. A time kinetics was also performed with the ratio of 4.25:1 (w/w) (vN�CRNA:chymotrypsin) in 10?mM Tris�CHCl, pH 8.0 at 37?��C from 0 to 20?min at different time intervals. Sitaxentan 100??g of vN�CRNA template was digested with chymotrypsin at a ratio of 4.25:1 to generate v?N�CRNA complex and then was diluted in 4?ml of TE buffer containing 0.1% (w/v) Triton X-100 and CsCl to give an initial density of 1.29?g/cc, and centrifuged to equilibrium in a Sorvall TH-660 rotor at 120,000?��?g for 16?h at 20?��C. The fractions containing the v?N�CRNA were diluted eightfold with the high salt buffer, HSB (1600?mM NaCl, 40?mM Tris�CHCl [pH 8.0], and 2?mM DTT) and then centrifuged at 120,000?��?g for 1?h in Sorvall TH-660 rotor. The resulting pellet of the v?N�CRNA was resuspended in TE buffer containing 10% glycerol and was dialyzed against the same buffer and then concentrated using Amicon Ultra-15 centrifugal filter device (Millipore) and stored at ??80?��C freezer. The purified vN�CRNA and v?N�CRNA protein bands were cut out from the gel, divided into smaller pieces, destained and washed with water and dehydrated in acetonitrile. The protein bands were then alkylated with iodoacetamide and digested in-gel by adding 5??l of Lenvatinib mw 20?ng/?l trypsin in 50?mM (NH4)2CO3 and incubated overnight selleckchem at room temperature. The resulting peptides were extracted from the polyacrylamide in two aliquots of 30??l 50% acetonitrile with 5% formic acid. These extracts were combined and evaporated to