1. Simple and efficient identification of chromatin modifying complexes and characterization of complex composition.
- Author
-
Lee JH and Skalnik D
- Subjects
- Centrifugation, Density Gradient methods, Chromatin Assembly and Disassembly, Chromatography, Affinity, HEK293 Cells, Humans, Mass Spectrometry methods, Molecular Sequence Annotation methods, Multiprotein Complexes isolation & purification, Protein Methyltransferases chemistry, Protein Methyltransferases isolation & purification, Protein Subunits isolation & purification, RNA Polymerase II chemistry, RNA Polymerase II isolation & purification, Receptors, Neuropeptide Y chemistry, Receptors, Neuropeptide Y isolation & purification, Recombinant Fusion Proteins, Multiprotein Complexes chemistry, Protein Subunits chemistry
- 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.
- Published
- 2013
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