Incorporating DNA shearing in standard affinity purification allows simultaneous identification of both soluble and chromatin-bound interaction partners.

ABSTRACT: Affinity purification coupled to mass spectrometry (AP-MS) is an effective means of identifying protein-protein interactions to better understand biological functions. However, issues associated with sample preparation still limit the success of AP-MS for specific classes of proteins, including those associated with chromatin that exhibit overall poor solubility in the protocols normally used for AP-MS analysis. Here, we wanted to provide a generally applicable method to simultaneously identify interactors for the chromatin-bound and the soluble fractions of a given bait protein. Using four FLAG-tagged canonical histone proteins (H2A, H2B, H3.1 and H4) we demonstrate that the chromatin solubility issue can be robustly alleviated by fragmenting DNA prior to AP-MS using a combination of soni

SUBMITTER: Lambert JP 

PROVIDER: S-EPMC4128382 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature