Affinity isolation of RNA polymerase II on amanitin-Sepharose.

We report here the first case of an affinity isolation of eukaryotic RNA polymerase II. The procedure employs an affinity matrix composed of alpha-amanitin coupled to Sepharose 4B via a ten atom spacer. RNA polymerase II from either calf thymus or wheat germ binds to the amanitin-Sepharose, as indicated by subsequent elution with sodium dodecylsulfate-containing buffer and analysis by polyacrylamide gel electrophoresis. The specificity of binding is demonstrated by the fact that when the enzyme is preincubated with 1 microgram/ml of free alpha-amanitin, subsequent binding to the amanitin-Sepharose is abolished. Elution methods that should permit the recovery of active enzyme from the column are discussed.