Absolute Quantitation of 2-D Protein Spots

Proteome analysis has been revolutionized by the marriage of two-dimensional (2-D) gel electrophoresis and mass spectrometry (1 –4 ). Mass spectrometry in combination with database searching permits the large-scale identification of proteins, and 2-D gel electrophoresis allows for the large-scale visualization and separation of cellular proteins. Previously, spot quantitation has been difficult, and numbers obtained were usually comparative, but not absolute (5 –7 ). Mass spectrometry and 2-D gel electrophoresis in tandem can be used to quantitate protein spots. This chapter covers protocols to quantitate precisely the relative amounts of protein observed in the 2-D gels using radiolabeled total cell extracts.