Tricine–SDS-PAGE.pdf

PROTOCOL Tricine–SDS Hermann Schägger Molekulare Bioenergetik, Zentrum der Biologischen Chemie, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, Haus 26, D-60590 Frankfurt, Germany. Correspondence should be addressed to H.S. (schagger@zbc.kgu.de). Published online 12 May 2006; doi:10.1038/nprot.2006.4 Tricine–SDSis commonly used to separate proteins in the mass range 1–100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate electroblotting, which is particularly crucial for hydrophobic proteins. Tricine–SDSis also used preferentially for doubled SDS(dSDS), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second dimension after blue-native PAGE (BN) and clear-native PAGE (CN). Here I describe a protocol for Tricine–SDS, which includes efficient methods for Coomassie blue or silver staining and electroblotting, thereby increasing the versatility of the approach. This protocol can be completed in 1–2 d. INTRODUCTION Glycine–SDS(also known as Laemmli–SDS)1 and because the stacking limit in the Laemmli system is too high, Tricine–SDS,3, based on glycine-Tris and Tricine-Tris and small proteins usually appear as smearing bands near the buffer systems, respectively, are the commonly used SDS electro- gel front. In a less convenient way, however, the small protein phoretic techniques for separating proteins. The acrylamide gels and peptide range can be accessed by making use of gradient gels used are often characterized by the total percentage concentration that continuously destack proteins according to dec