Small-scale density gradient sedimentation to separate and analyze multiprotein complexes.
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.
Tanese
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Department of Microbiology, New York University Medical Center, New York 10016, USA. tanesn01@mcrcr.med.nyu.edu
D002499:Centrifugation, Density Gradient / Q000379:methods*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification*; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification; D019473:Transcription Factors, TFII / Q000737:chemistry / Q000302:isolation & purification
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII