tRNA was radiolabeled at the final bridging phosphate with α-32P-ATP and E. coli CCA adding enzyme as previously described (16 (link)). Briefly, 40 pmol E. coli tRNATyr (Sigma-Aldrich) was renatured in 20 mM Tris–HCl, pH 7.0 by heating to 85°C for 2 min followed by cooling at room temperature for 3 min. MgCl2 was added to 10 mM, and the tRNA was incubated at 37°C for 5 min. Renatured tRNA was then treated with recombinant, purified E. coli CCA adding enzyme (final concentration = 30 μg/ml) at 37°C for 10 min in 50 mM glycine, pH, 9.0, 13 mM MgCl2, 50 μM sodium pyrophosphate, 20 μM cytidine triphosphate (CTP), 0.33 μM α-32P-ATP. Labeled tRNA was purified by denaturing polyacrylamide gelelectrophoresis (PAGE) (7M Urea, 1× Tris-Borate-EDTA (TBE) Buffer).
E coli trnatyr
Manufactured by Merck Group
Sourced in United States
E. coli tRNATyr is a laboratory-produced sample of transfer RNA (tRNA) molecules specific to the amino acid tyrosine, derived from the bacterium Escherichia coli. It serves as a tool for researchers to study the structure, function, and interactions of tRNA in biological processes.
Automatically generated - may contain errors
2 protocols using e coli trnatyr
1
Radiolabeling of tRNA with CCA Enzyme
2
Purification of Ribonuclease Cusativin
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Escherichia coli tRNA and RNase T1 were procured from Roche Diagnostics (Indianapolis, IN, USA). E. coli tRNATyr was obtained from Sigma-Aldrich (St. Louis, MO, USA). Riboflavin and all other chemicals were acquired from Fisher Scientific (Fairlawn, NJ, USA) unless otherwise specified. Ribonuclease cusativin was purified from the overexpression strain of E. coli on a nickel column using a His-tag protein purification kit from EMD Millipore (Burlington, MA, USA) as described [51 (link)].
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