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Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes
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Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

Article DOI: 10.3791/51720-v • 08:44 min • October 23rd, 2014
October 23rd, 2014 •
1,2, 1, 1,2, 1,2
1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center

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Summary
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This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Tags

Generation Purification Human INO80 Chromatin Remodeling Complexes Subcomplexes Nucleosome Dynamics DNA Accessibility ATP-dependent Nucleosome Remodeling Protein Subunits Ino80 SNF2-like ATPase Scaffold Assembly Subunit Functions Chromatin Remodeling Activity INO80 Subassemblies Human Cells Heterologous Expression Systems JOVE Protocol Purification Procedure N-terminally FLAG Epitope Tagged Ino80 CDNA Human Embryonic Kidney (HEK) 293 Cell Lines Flp-mediated Recombination Mutant Ino80 Proteins Subunit Deletion SiRNAs Targeting Subunit FLAG Immunoprecipitation Protein Fractions
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