title: Preparation of nuclear and cytoplasmic extracts from mammalian cells.
abstract: Extracts prepared from the isolated nuclei of cultured cells have been instrumental in dissecting the mechanisms by which transcription and mRNA processing occur. These extracts are able to recapitulate accurate transcription initiation and splicing in vitro, which has been useful in direct functional studies. They also serve as the starting material for purification of proteins that can then be reassembled in functional studies or examined in more detail biochemically. This unit describes the preparation of nuclear extracts from cultured cells and optimized production of transcriptionally active extracts from HeLa cells. Additional protocols describe optimization of the method to increase the yield of specific proteins, adaptation of the method for downstream applications such as affinity purification, and preparation of the cytoplasmic (S-100) fraction.
authors.affiliation: The Stowers Institute for Medical Research, Kansas City, Missouri, USA.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Abmayr
authors.forename: Tingting
authors.initials: T
authors.lastname: Yao
authors.forename: Tari
authors.initials: T
authors.lastname: Parmely
authors.forename: Jerry L
authors.initials: JL
authors.lastname: Workman
mesh_terms: D000818:Animals; D002457:Cell Extracts / Q000737:chemistry*; D002467:Cell Nucleus / Q000737:chemistry*; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D003593:Cytoplasm / Q000737:chemistry*; D003600:Cytosol / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D006367:HeLa Cells; D006801:Humans; D008322:Mammals; D014157:Transcription Factors; D014158:Transcription, Genetic
chemical_list: D002457:Cell Extracts; D004268:DNA-Binding Proteins; D014157:Transcription Factors