Large-Scale Immunoprecipitation from Fission Yeast Cell Extracts.
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.
CRUK Cell Division Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester M20 4BX, United Kingdom Agnes.Grallert@cruk.manchester.ac.uk Iain.Hagan@cruk.manchester.ac.uk.
Agnes
A
Grallert
CRUK Cell Division Group, Cancer Research UK Manchester Institute, University of Manchester, Manchester M20 4BX, United Kingdom Agnes.Grallert@cruk.manchester.ac.uk Iain.Hagan@cruk.manchester.ac.uk.
Iain M
IM
Hagan
D011232:Chemical Precipitation; D045424:Complex Mixtures / Q000737:chemistry*; D005656:Fungal Proteins / Q000302:isolation & purification*; D047468:Immunoprecipitation / Q000379:methods*; D013058:Mass Spectrometry; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D012568:Schizosaccharomyces / Q000737:chemistry*; D014238:Trichloroacetic Acid / Q000378:metabolism
D045424:Complex Mixtures; D005656:Fungal Proteins; D014238:Trichloroacetic Acid