Proteome analysis with classical 2D-PAGE.
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is based on the combination of two orthogonal separation techniques. In the first dimension, proteins are separated by their isoelectric point, a technique known as isoelectric focusing (IEF). There are two important variants of IEF, which are carrier-ampholine (CA)-based IEF and immobilized pH gradient (IPG)-based IEF. In the second dimension, proteins are further separated by their electrophoretic mobility using SDS-PAGE. Finally, proteins can be visualized and quantified by different staining procedures, such as Coomassie, silver, or fluorescence staining. This chapter gives detailed protocols for 2D-PAGE, using both CA- and IPG-based separation in the first dimension.
Department of Medical Proteomics/Bionalaytics, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany. caroline.may@rub.de
Caroline
C
May
Frederic
F
Brosseron
Kathy
K
Pfeiffer
Helmut E
HE
Meyer
Katrin
K
Marcus
D000665:Ampholyte Mixtures / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007525:Isoelectric Focusing; D020543:Proteome / Q000302:isolation & purification*; D018892:Proton-Motive Force
D000665:Ampholyte Mixtures; D020543:Proteome