The current state of two-dimensional electrophoresis with immobilized pH gradients.
The original protocol of two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt; Gorg et al., Electrophoresis 1988, 9, 531-546) is updated. Merits and limits of different methods for sample solubilization, sample application (by cup-loading or ingel rehydration) with respect to the pH interval used for IPG-isoelectric focusing are critically discussed. Guidelines for running conditions of analytical and micropreparative IPG-Dalt, using wide IPGs up to pH 12 for overview patterns, or narrow IPGs for zoom-in gels for optimum resolution and detection of minor components, are stated. Results with extended separation distances as well as automated procedures are demonstrated, and a comparison between protein detection by silver staining and fluorescent dyes is given. A brief trouble shooting guide is also included.
Görg
A
A
Proteomics Group, Institute of Food Technology and Analytical Chemistry, Technical University of Munich, Freising-Weihenstephan, Germany. angelika.gorg@lrz.tum.de
Obermaier
C
C
Boguth
G
G
Harder
A
A
Scheibe
B
B
Wildgruber
R
R
Weiss
W
W
D000818:Animals; D001331:Automation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes; D005782:Gels; D006863:Hydrogen-Ion Concentration; D011506:Proteins / Q000032:analysis*; D016622:Silver Staining / Q000379:methods; D012967:Sodium Dodecyl Sulfate
D005456:Fluorescent Dyes; D005782:Gels; D011506:Proteins; D012967:Sodium Dodecyl Sulfate