Benzoylated diethylaminoethyl cellulose chromatography of tumor and nontumor transfer RNA

M W Taylor
1970 Cancer Research  
run at 4°. Packed columns were washed with 2 M NaCl solution until no further material absorbing at 260 m@.t was eluted and then equilibrated with 0.4 M NaCl solution, 0.01 M MgCl2 , and 0.05 M sodium acetate, pH 5 .0. All subse quent solutions were made up in sodium acetate buffer, pH 5.0, and 0.01 M MgCl2 . Samples of tRNA were loaded onto the column in 0.4 M NaCl solution. Unacylated tRNA was eluted with a linear gradient of 500 ml of 0.4 M NaC1 solution and 500 ml of 1.0 M NaCl solution.
more » ... flow rate was 0.5 nil/nun. At the completion of this gradient, 100 ml of 1 M NaCl, containing 10% ethanol, were washed through the column. Fractions of 10 ml were collected, concentrated, and acylated as described below. Acylated tRNA was chromatographed with 5 mg of E. coli tRNA (Schwarz BioResearch, Inc., Orangeburg, N. Y.) as carrier. The elution conditions used depended on the species of tRNA, and are described in the respective figures. Fractions were collected, precipitated with alcohol, air dried, and counted as described previously (19) . Amino Acid Acceptor Activity. One-mi aliquots from each column fraction were withdrawn and precipitated with 3 ml 95% alcohol in the presence of glycogen. The precipitate was resuspended after drying in 0.1 ml sterile water. To this was added 0.05 ml of a reaction mixture containing 0.04 M MgCl, 0.02 M sodium cacodylate buffer (pH 7.4), 2 X i0@ M 19 amino acids excepting the labeled amino acids 0.1 pCi
pmid:5528921 fatcat:6pzi2bikxvg23j3bjpczht4bgi