Micro bio spin columns with bio gel p 30

Gel shift assays were performed^{24 (link)}. Gel shift assays for observation of reactivity were performed with 500 μM BXO per sample unless otherwise noted. Aminoacylated RNA was incubated with 95 nmol streptavidin and run on an 8% native polyacrylamide gel with 0.5X TBE. For determining the uncatalyzed reaction rate with BFO, aminoacylation reactions were performed in 50 μL selection buffer with 5% acetonitrile containing BFO at 1250, 250, 50, 10, or 2 μM and 0.43 μM random library RNA which was fluorescently labeled using 5’ EndTag Nucleic Acid Labeling System (Vector Laboratories) and fluoroscein maleimide (TCI Chemicals). Reactions were incubated at room temperature for 90 minutes with rotation and stopped by desalting using Micro Bio-Spin Columns with Bio-Gel P-30 (Bio-Rad Laboratories). 95 nmol of streptavidin (New England Biolabs) was added to each sample, which were then incubated for 15 minutes with rotation at room temperature, run on an 8% polyacrylamide gel, imaged on an Amersham Typhoon 5 Biomolecular Imager, and analyzed using ImageQuant 8.1 software. For uncatalyzed reaction rate determination, all high molecular weight bands were grouped and compared to total RNA quantified in the lane to calculate the fraction reacted at each concentration, which was fit to the kinetic model.