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Published on: May 3, 2015
Large-Scale Immunoprecipitation from Fission Yeast Cell Extracts
Agnes Grallert1, Iain M Hagan1
1CRUK 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.
This study presents immunoprecipitation (IP) methods to isolate molecules for posttranslational modification (PTM) analysis. Procedures preserve PTMs during isolation, enabling mass spectrometry identification of modifications and associated proteins.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Posttranslational modifications (PTMs) are crucial for protein function and cellular signaling.
- Identifying PTMs requires isolating sufficient quantities of target molecules.
- Existing methods may alter PTM profiles during sample preparation.
Purpose of the Study:
- To develop and present robust immunoprecipitation (IP) protocols for isolating molecules of interest.
- To enable accurate identification of posttranslational modifications (PTMs) using targeted mass spectrometry.
- To preserve PTM integrity throughout the isolation process.
Main Methods:
- Immunoprecipitation (IP) procedures for molecule isolation.
- In situ denaturation using trichloroacetic acid (TCA) precipitation.
- Nondenaturing IP variation for preserving molecular interactions.
Main Results:
- Established IP protocols yield sufficient quantities of target molecules for PTM analysis.
- In situ denaturation effectively inhibits modifying enzymes, preserving endogenous PTMs.
- Nondenaturing IP allows mapping of PTMs on associated proteins, with potential modifications during IP.
Conclusions:
- The described IP methods are effective for isolating molecules and preserving PTMs for mass spectrometry.
- The choice between denaturing and nondenaturing IP depends on whether PTMs on associated proteins are of interest.
- These protocols facilitate comprehensive PTM analysis and the study of protein signaling networks.
