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[15] Sepharose chromatography of transfer ribonucleic acids using reverse salt gradients

1979; Academic Press; Linguagem: Inglês

10.1016/0076-6879(79)59084-3

1557-7988

G. Wesley Hatfield,

DNA and Nucleic Acid Chemistry

This chapter describes the use of column chromatography method designed specifically for the separation of tRNA molecules, Sepharose chromatography using reverse salt gradients. This method involves the use of high concentrations of an antichaotropic ion, such as the phosphate or sulfate anions, to induce selective binding of the tRNA molecules to Sepharose beads. Although the exact nature of the binding and fractionation of tRNA on these columns is not completely understood, it has been suggested that these interactions may be, at least in part, hydrophobic in nature. Either the unaminoacylated or the [3H] aminoacylated tRNA sample is applied to the equilibrated Sepharose column. The sample is eluted from the column at 4° with a decreasing linear (NH4)2SO4 gradient using 234 ml of 1.3 M (NH4)2SO4 in buffer I in the mixing chamber and 250 ml of buffer I [containing no (NH4)2SO4] in the reservoir. The column is allowed to flow at a rate of 40 ml/hr. If unaminoacylated tRNA is chromatographed, the fractions are assayed for specific tRNA species using the column effluent fractions aminoacylation assay.