Chapter 10. 3D-Gel Electrophoresis – A New Development in Protein Analysis

  1. Haroun N. Shah and
  2. Saheer E. Gharbia
  1. Robert Ventzki1 and
  2. Josef Stegemann2

Published Online: 15 JUN 2010

DOI: 10.1002/9780470665497.ch10

Mass Spectrometry for Microbial Proteomics

Mass Spectrometry for Microbial Proteomics

How to Cite

Ventzki, R. and Stegemann, J. (2010) 3D-Gel Electrophoresis – A New Development in Protein Analysis, in Mass Spectrometry for Microbial Proteomics (eds H. N. Shah and S. E. Gharbia), John Wiley & Sons, Ltd, Chichester, UK. doi: 10.1002/9780470665497.ch10

Editor Information

  1. Department for Bioanalysis and Horizon Technologies, Health Protection Agency Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK

Author Information

  1. 1

    Am Neuberg 2, D-69221 Dossenheim, Germany

  2. 2

    Kühler Grund 50, 69126 Heidelberg, Germany

Publication History

  1. Published Online: 15 JUN 2010
  2. Published Print: 23 JUL 2010

ISBN Information

Print ISBN: 9780470681992

Online ISBN: 9780470665497



  • 3D-gel electrophoresis;
  • comparative protein analysis;
  • large scale screening;
  • online detection;
  • laser induced fluorescence


Three-dimensional (3D)-gel electrophoresis is a new method for protein analysis in a separation medium that extends substantially in all three spatial dimensions. Proteins can be analyzed according to one, two, or three independent separation parameters, i.e., native size, pI, and molecular mass (MM). As the samples are applied in a two-dimensional array to the top surface of a 3D-geometry gel body, the method provides a very high sample capacity: For separation according to one parameter, the 3D-gel can accommodate up to 1536 samples loaded directly from a microtiter plate (MTP). For two-parameter separation, up to 36 immobilized pH gradient (IPG) strips containing samples pre-separated by isoelectric focusing (IEF) can be loaded. Sample migration and separation occur along the third spatial dimension, perpendicular to the loading surface. In this way, all samples are analyzed in the same gel under identical electrophoretic and thermal conditions, thus avoiding gel-to-gel variations and significantly increasing the efficiency of the process. Labeled proteins are detected online by laser-induced fluorescence (LIF). Images are acquired by a digital camera and electronically recorded as a 3-D image stack during electrophoresis. Image processing software generates vertical sections of the 3-D image stack, mimicking a series of conventional slab gel scans and making results immediately available without further gel processing. The direct comparability of separation patterns allows the introduction of a third parameter to two-dimensional electrophoresis (2-DE) analysis by extracting protein samples from cells as a series with increments in time, concentration or dosage regimen. With the sample index n (n=1…36) denoting this variable, the 3D-gel method quantitatively measures the protein abundance as a function of the respective parameter. This procedure makes the method a valuable tool for large scale comparative proteomics studies with a wide range of applications in molecular discovery, clinical diagnosis, pharmacology, and toxicology.