Identifying chromatin readers using a SILAC-based histone peptide pull-down approach.
Posttranslational modifications (PTMs) on core histones regulate essential processes inside the nucleus such as transcription, replication, and DNA repair. An important function of histone PTMs is the recruitment or stabilization of chromatin-modifying proteins, which are also called chromatin "readers." We have developed a generic SILAC-based peptide pull-down approach to identify such readers for histone PTMs in an unbiased manner. In this chapter, the workflow behind this method will be presented in detail.
Molecular Cancer Research, University Medical Center Utrecht, 3584 CG, Utrecht, The Netherlands. m.vermeulen-3@umcutrecht.nl
Michiel
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Vermeulen
D000818:Animals; D018929:Cell Culture Techniques; D002457:Cell Extracts / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006657:Histones / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D007553:Isotope Labeling; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D014357:Trypsin / Q000737:chemistry
D002457:Cell Extracts; D002843:Chromatin; D006657:Histones; D010446:Peptide Fragments; D014357:Trypsin