A ribonuclease assay: separation of product from undegraded aminoacyl-transfer RNA substrate.
A new rapid microassay for RNase is based on the extraction of the products of the degradation of aminoacyl-tRNA into ethyl acetate. The substrate, for example, f[3H]Met-tRNA, is not soluble in ethyl acetate but the product f[3H]Met-nucleotide is extracted with high efficiency over a wide range of pH in phosphate buffer of high ionic strength. The limit digest of f[3H]Met-tRNA by RNase T1 in which the minimum-sized product should be f[3H]Met-hexanucleotide is also soluble in ethyl acetate. The assay is sensitive to 0.05 pg of RNase A, has a very low background, and is linear with enzyme concentration. The most economic substrate is unfractionated tRNA containing a small concentration of the radiolabeled aminoacyl-tRNA.
D000085:Acetates; D007700:Kinetics; D012346:RNA, Transfer, Amino Acyl / Q000032:analysis*; D012260:Ribonucleases / Q000302:isolation & purification*
D000085:Acetates; D012346:RNA, Transfer, Amino Acyl; C007650:ethyl acetate; D012260:Ribonucleases