Smithies and Poulik first proposed to use 2D electrophoresis to separate proteins in a much better resolution than traditional one dimensional electrophoresis [1]. Their multidimensional principle of complex proteome separation was also adopted by chromatography and capillary electrophoresis protein separations. In stead of using the cylindrical tube gels, in 1964 Raymond demonstrated the use of the flat slab polyacrylamide gels which allow the two-dimensional separation of proteins [2].

In 1975 O'Farrell introduced 2D-PAGE electrophoresis to separate cellular protiens under denaturing conditions [3]. O'Farrell's method utilized isoelectric focusing (IEF) as the first dimension protein separation. Proteins are first separated in 1D IEF according to their isolectric point and then followed by a second dimension SDS-PAGE electrophoresis which separates proteins according to their molecular weight. With the introduction of immobilized pH gradients (IPGs) by Bjellqvist et al., first dimensional separation by IEF yields higher resolution as proteins with similar isoelectric point (pI) can now be separated using IPG-IEF [4]. After 2D-PAGE electrophoresis, gel images ca be acquired by imaging system for further spot detection, normalization, protein profiling, background correction before final data analysis.

Basic 2D-PAGE electrophoresis procedure:

1. Sample preparation
2. IPG strips and SDS gel casting
3. IEF in IPG strips first dimensional separation
4. Equilibration of IPG strips after first dimension electrophoresis
5. Second dimension SDS-PAGE separation
6. Gel staining and visualization
7. Blotting
8. Protein spots characterization
9. Data analysis

2D-PAGE gel electrophoresis protocols

1. Protein gel electrophoresis and 2D-gel
2. 2D-PAGE gel electrophoresis protocol
3. 2D-gel protocol
4. 2D gel electrophoresis Immobilized pH gradient (IPG) strips

2-D electrophoresis Analysis Software resources

• Nonlinear Dynamics: http://www.nonlinear.com/
• PDQuest: http://proteomeworks.bio-rad.com/html/tech5.html
• Flicker for 2D gel analysis: http://www-lecb.ncifcrf.gov/flicker/
• NCI/NCRDC LMMB Image Processing Section (GELLAB software): http://www-lecb.ncifcrf.gov/lemkin/gellab.html
• Compugen (Z3 software): http://www.2Dgels.com
• Expasy Index to 2D PAGE databases and services: http://www.expasy.ch/ch2d/2d-index.html
• HSC 2DE Gel Protein Databases list: http://www.harefield.nthames.nhs.uk/nhli/protein/other_sites.html
• Phosphoprotein Database: http://www-lecb.ncifcrf.gov/phosphoDB/
• Rat Serum Protein Database: http://linux.farma.unimi.it
• Parasite Proteome Maps: http://www.aber.ac.uk/~mpgwww/Proteome/ProtMaps.html
• Cambridge Proteomics Facility: http://www.bio.cam.ac.uk/proteomics/index.html
• Rice 2D Database: http://semele.anu.edu.au/2d/2d.html
• COMPLUYEAST-2D PAGE Database: http://babbage.csc.ucm.es/2d/2d.html
• Danish Centre for Human Genome Research 2D PAGE Databases (Aarhus): http://proteomics.cancer.dk
• Siena-2DPAGE: http://www.bio-mol.unisi.it/2d/2d.html
• PMMA-2D Page—at Purkyne Military Medical Academy, Czech: http://www.pmma.pmfhk.cz/
• Human Colon Carcinoma Protein Database-Joint ProteomicS Laboratory (JPAL), Ludwig Institute for Cancer Research, Melbourne, Austrailia: http://www.ludwig.edu.au/jpsl/jpslhome.html
• HP-2D PAGE (Max Delbruck Center, Berlin): http://www.mdc-berlin.de/~emu/heart
• MitoDat—Mendelian Inheritance and the Mitochondrion: http://www-lmmb.ncifcrf.gov/mitoDat/
• SWISS-2DPAGE at Geneva University Hospital: http://www.expasy.ch/ch2d/ch2d-top.html
• Proteome BioKnowledge® Library: http://www.proteome.com/YPDhome.html
• Yeast Proteome Map: http://www.ibgc.u-bordeaux2.fr/YPM/
• Melanie: http://us.expasy.org/melanie

References:

• Smithies O. Poulik D. M., Two-dimensional electrophoresis of serum proteins. Nature. 1956 Jun 2;177(4518):1033
• Raymond S., Acrylamide gel electrophoresis, Ann. N. Y. Acad. Sci., P350 - P365
• O'Farrell H. P., High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem., P4007 - P4021
• Bjellqvist B. Ek K. Righetti G. P. Gianazza E. Gorg A. Westermeier R. Postel W., Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications, J. Biochem. Biophys. Methods, P317 - P339
• 2D-PAGE electrophoresis online overview and manual.(Prof. Dr. Dr. Angelika Görg)