2-Dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE)

In 2-D PAGE protein complements of two or more different samples are compared by their relative staining intensities after in-gel detection of proteins e.g. by fluorescent dyes or colloidal coomassie. Differences between two samples are excised from the gel and in-gel digested with a site-specific protease such as trypsin. The tryptic peptides are then extracted from the gel and subjected to tandem mass spectrometry (usually MALDI TOF/TOF MS) for protein identification.

Alternatively a 2-D PAGE/MALDI TOF/TOF MS experiment can be extended to a large scale approach to create a 2-D protein reference map. Therefore all visualise protein spots are excised and subjected to mass spectrometry-based protein identification. The reference map is then used to compare protein abundances in various gels of different related samples by matching the positions of protein spots to the identified proteins of the reference map without any further need for MS-based protein identification. This approach gives a more comprehensive picture of the relative protein abundances e.g. of whole pathways or protein complexes.