Isolation of the Mediator–pol II–TFIIF assembly. (A) Schematic of the protocol. The gradient was set up such that the complete Mediator–pol II–TFIIF assembly would migrate and concentrate in the final fraction. Note that Mediator is not bound to an activator in these experiments. (B) Silver-stained gel of the glycerol gradient fractions. Free TFIIF, free Mediator, and free pol II migrate earlier in the gradient, as indicated. Subunit identities shown at the right. (C) Western blot analysis confirms the presence of Mediator, pol II, and TFIIF in the final gradient fraction, as expected. The pol II antibody was directed against RPB1, the Mediator antibody targeted MED23, and the TFIIF antibody RAP74.