title: Preparation of Drosophila Kc cell nuclear extracts for in vitro splicing.
abstract: This protocol describes how to prepare nuclear extracts from cultured Drosophila cells. It uses the Kc cell line, which grows without fetal bovine serum and so is suitable for large-scale suspension cultures. The suspension cells are harvested by centrifugation, washed to remove media components, and resuspended in a low-salt (hypotonic) buffer that makes the cells swell in size and easy to homogenize. Usually, nuclear extract preparations are performed on 4-32 L of cells at ∼ 3.5 × 10(6) cells/mL.
authors.forename: Donald C
authors.initials: DC
authors.lastname: Rio
mesh_terms: D000818:Animals; D002457:Cell Extracts / Q000302:isolation & purification*; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D003584:Cytological Techniques / Q000379:methods*; D004330:Drosophila / Q000201:enzymology*; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D012261:Ribonucleoproteins / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D002457:Cell Extracts; D012322:RNA Precursors; D012261:Ribonucleoproteins