Small-scale density gradient sedimentation to separate and analyze multiprotein complexes

Abstract

The transcription factor TFIID is a multisubunit complex that is required for promoter recognition and accurate initiation of transcription by RNA polymerase II. To dissect the molecular architecture and the biochemical properties of TFIID, a small-scale density gradient sedimentation method is employed to separate related complexes through differences in their sedimentation properties. A small amount of starting material is sufficient to obtain readily assayable amounts of separated proteins after centrifugation for 8 to 12 h in a benchtop ultracentrifuge. Gradient fractions are analyzed by immunoblotting for the presence of specific components of TFIID. Sucrose gradient sedimentation is performed to separate a mixture of multiprotein complexes from a crude nuclear extract. Immunoprecipitation of the proteins present in each fraction with an anti-TBP antibody reveals multiple TBP-containing complexes of different sizes. Density gradient sedimentation permits separation of specific components in a complex mixture and preserves activity, allowing functional assays.