INTRODUCTIONThis protocol describes a method for in-gel digestion of proteins after fractionation using SDS-PAGE. It is applicable to both one- and two-dimensional polyacrylamide gels of different thicknesses, acrylamide concentrations, and band (spot) sizes. This protocol followed by liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) or matrix-assisted laser desorption/ionization/time of flight/time of flight (MALDI-TOF/TOF) is capable of identifying low femtomole quantities of a protein. High-sensitivity mass spectrometry should be capable of identifying attomole quantities of protein.