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Sample Preparation and Protein Determination for 2D-DIGE Proteomics.

Stephen GarganKay Ohlendieck
Published in: Methods in molecular biology (Clifton, N.J.) (2022)
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.
Keyphrases
  • protein protein
  • molecularly imprinted
  • mass spectrometry
  • binding protein
  • solid phase extraction
  • quantum dots
  • microbial community
  • label free
  • living cells