2.7. tRNA binding
Binding of PTRHD1 to free and peptidyl-tRNA was carried out in
15 mL volumes composed of 8.4 mg tRNA in 7 mL, 4.75e8 mL ddH2O
(depending on the volume of PTRHD1 added), and 0e3.25 mL of
360 mM PTRHD1. Free tRNA was prepared by cleaving peptidyltRNA
with E. coli Pth1 for an extended duration. The PTRHD1
mixture was incubated for 20 min at room temperature before
10 mL aliquots were run on a 1.2% agarose gel at 90 V for ~30 min at
room temperature. Gels were stained with ethidium bromide for
visualization.