Multifluorescence 2D Gel Imaging and Image Analysis - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 21

Chapter 3
Multifl uorescence 2D Gel Imaging and Image Analysis
Ingo Vormbrock , Sonja Hartwig , and Stefan Lehr
Abstract
Although image acquisition and analysis are crucial steps within the multifl uorescence two-dimensional
gel electrophoresis workfl ow, some basics are frequently not carried out with the necessary diligence. This
chapter should help to prevent easily avoidable failures during imaging and image preparation for com-
parative protein analysis.
Key words: 2DE, Two-dimensional gel electrophoresis, Cyanine fl uorescence dyes, Gel imaging,
Image analysis, 2DE Multifl uorescence analysis, Multifl uorescence analysis, Protein visualization,
Spot detection, DIGE
1. Introduction
Multifl uorescence two-dimensional gel electrophoresis (2DE) is
theoretically capable of detecting and quantifying thousands of
protein spots in a single gel with a detection limit below 0.5 fmol
of protein and a dynamic range of up to fi ve orders of magnitude.
Enormous volumes of high-quality data can be derived from multi-
fl uorescence 2DE gels provided that image acquisition, processing
and analysis are performed according to optimized and standardized
operating protocols. Here we describe the instrumentation and the
most critical parameters for highly sensitive and reproducible fl uo-
rescence imaging, spot detection and quantifi cation.