The identification of tRNA isoacceptors fractionated by polyacrylamide gel electrophoresis.
A method is described by which specific tRNA isoacceptors may be identified in small amounts of bulk tRNA. The strategy relies on the retention of aminoacyl-tRNA by CNBr-Sepharose through covalent coupling of the alpha-NH2 group of the amino acid to the matrix. After removing unbound material by thorough washing, the bound specific isoacceptors are released by cleavage of the labile aminoacyl-tRNA ester bond through mild alkaline treatment. The product is analysed by two-dimensional gel electrophoresis and the spots obtained may be correlated with the pattern from bulk tRNA. Optimum sensitivity is achieved by combining the method with the recently introduced silver staining technique (Igloi, G.L. (1983) Anal. Biochem. 134, 184-188) for tRNA.
D000596:Amino Acids / Q000378:metabolism; D003488:Cyanogen Bromide / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D012329:RNA, Bacterial / Q000302:isolation & purification; D012346:RNA, Transfer, Amino Acyl / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000378:metabolism; D013997:Time Factors
D000596:Amino Acids; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D012685:Sepharose; D003488:Cyanogen Bromide