Large-scale immunoprecipitation from fission yeast cell extracts.

Abstract

We outline immunoprecipitation (IP) procedures to isolate the large quantities of a molecule of interest that are required to identify posttranslational modifications (PTMs) in subsequent targeted mass spectrometry analysis. In situ denaturation by trichloroacetic acid precipitation inhibits the activities of modifying enzymes that could alter the PTM profile to preserve the PTMs on a target of interest throughout the precipitation step. In contrast, isolation of the same molecule with the nondenaturing variation on this IP procedure can maintain associations with partner molecules whose PTMs can also be mapped, albeit with the caveat that modifications could have occurred during the extended IP period.