Sample Preparation and Protein Determination for 2D-DIGE Proteomics

Stephen Gargan; Kay Ohlendieck

doi:10.1007/978-1-0716-2831-7_22

Sample Preparation and Protein Determination for 2D-DIGE Proteomics

Methods Mol Biol. 2023:2596:325-337. doi: 10.1007/978-1-0716-2831-7_22.

Authors

Stephen Gargan¹, Kay Ohlendieck²

Affiliations

¹ Department of Biology, Maynooth University, National University of Ireland, Maynooth, Co. Kildare, Ireland.
² Department of Biology, Maynooth University, National University of Ireland, Maynooth, Co. Kildare, Ireland. kay.ohlendieck@mu.ie.

PMID: 36378448
DOI: 10.1007/978-1-0716-2831-7_22

Abstract

Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a widely employed method for efficient protein separation and the determination of abundance changes in distinct proteoforms. This makes this gel-based method a key technique of comparative approaches in top-down proteomics. For the appropriate screening of proteome-wide alterations, initial preparative steps involve sample handling, homogenization, subcellular fractionation, and the determination of protein concentration, which makes the optimal application of these techniques a crucial part of a successful initiation of a new 2D-DIGE-based analysis. This chapter describes sample homogenization and a standardized protein assay for the preparation of homogenates with a known protein concentration for subsequent differential fluorescent tagging and two-dimensional gel electrophoretic separation.

Keywords: Difference gel electrophoresis; Homogenization; Protein assay; Proteomics; Top-down proteomics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Electrophoresis, Gel, Two-Dimensional / methods
Proteome*
Proteomics* / methods
Specimen Handling
Two-Dimensional Difference Gel Electrophoresis / methods

Substances

Proteome