Purification of recombinant Drosophila ACF.
The goal of chromatin assembly procedures is to prepare extended nucleosomal arrays from cloned DNA templates and purified core and linker histones. The assembled chromatin should be highly defined in its protein content and resemble bulk chromatin isolated from living cell nuclei in terms of periodicity and nucleosome positioning. This protocol describes the preparation of Drosophila ACF (ATP-utilizing chromatin assembly and remodeling factor) for use in chromatin assembly reactions. In this method, ACF is prepared by the coexpression of the carboxyl-terminally FLAG-tagged Acf1 subunit with the untagged ISWI subunit in baculovirus. The complex is then purified in one step by FLAG immunoaffinity chromatography. This procedure typically results in a stoichiometric complex of Acf1 and ISWI.
Peterson
Craig L
CL
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D007313:Insecta; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification*
C120419:Acf protein, Drosophila; D002843:Chromatin; D029721:Drosophila Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors