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

N Tanese
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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.

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Centrifugation, Density GradientDNA-Binding ProteinsHeLa CellsHumansMacromolecular SubstancesMultiprotein ComplexesNuclear ProteinsTATA-Box Binding ProteinTranscription Factor TFIIDTranscription FactorsTranscription Factors, TFIIJournal Article

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N Tanese. 1997. Small-scale density gradient sedimentation to separate and analyze multiprotein complexes.. https://doi.org/10.1006/meth.1997.0475

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