Tricine-SDS-PAGE.
Tricine-SDS-PAGE is 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-SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), 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-PAGE) and clear-native PAGE (CN-PAGE). Here I describe a protocol for Tricine-SDS-PAGE, 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.
Schägger
Hermann
H
Molekulare Bioenergetik, Zentrum der Biologischen Chemie, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, Haus 26, D-60590 Frankfurt, Germany. schagger@zbc.kgu.de
D020106:Acrylamide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D015336:Molecular Probe Techniques; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D012394:Rosaniline Dyes; D016622:Silver Staining
D011506:Proteins; D012394:Rosaniline Dyes; D020106:Acrylamide; C048139:Coomassie blue; D005998:Glycine; C100184:tricine