Two-dimensional difference gel electrophoresis

Methods Mol Biol. 2012;869:287-304. doi: 10.1007/978-1-61779-821-4_24.

Abstract

Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2DE) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as "spots" with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With the appropriate imaging system, difference gel electrophoresis (DIGE) is capable of reliably detecting as little as 0.2 fmol of protein, and protein differences down to ±15%, over a ∼20,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2-3 days to complete.

MeSH terms

  • Animals
  • Buffers
  • Carbocyanines / chemistry
  • Cell Extracts / chemistry
  • Cell Extracts / isolation & purification*
  • Fluorescent Dyes / chemistry
  • Humans
  • Image Processing, Computer-Assisted
  • Proteins / chemistry
  • Proteins / isolation & purification*
  • Staining and Labeling
  • Two-Dimensional Difference Gel Electrophoresis / methods*

Substances

  • Buffers
  • Carbocyanines
  • Cell Extracts
  • Fluorescent Dyes
  • Proteins
  • cyanine dye 3
  • cyanine dye 5