title: Simple and efficient identification of chromatin modifying complexes and characterization of complex composition.
abstract: Affinity purification and mass spectrometry analysis have been used to identify and characterize protein complexes. Wdr82-associated chromatin modifying complexes were purified by single-step FLAG affinity purification from human cells induced to express FLAG-tagged Wdr82. Purified proteins were analyzed by SDS-PAGE and specific protein bands were identified by mass spectrometry. Subsequently, purified proteins were fractionated on sucrose gradient equilibrium centrifugation to determine overall composition of each identified complex. We describe here simple and efficient approaches for the identification of chromatin modifying complexes and subsequent characterization of complex composition.
authors: Department of Pediatrics, Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.
authors: Jeong-Heon
authors: JH
authors: Lee
authors: David
authors: D
authors: Skalnik
mesh_terms: D002499:Centrifugation, Density Gradient / Q000379:methods; D042002:Chromatin Assembly and Disassembly; D002846:Chromatography, Affinity; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D058977:Molecular Sequence Annotation / Q000379:methods; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000302:isolation & purification; D011496:Protein Methyltransferases / Q000737:chemistry / Q000302:isolation & purification; D021122:Protein Subunits / Q000737:chemistry* / Q000302:isolation & purification; D012319:RNA Polymerase II / Q000737:chemistry / Q000302:isolation & purification; D017476:Receptors, Neuropeptide Y / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins
chemical_list: D046912:Multiprotein Complexes; D021122:Protein Subunits; D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; C109491:neuropeptide Y4 receptor; D011496:Protein Methyltransferases; D012319:RNA Polymerase II