A novel Bicine running buffer system for doubled sodium dodecyl sulfate - polyacrylamide gel electrophoresis of membrane proteins.
A novel, Bicine-based SDS-PAGE buffer system was developed for the analysis of membrane proteins. The method involves molecular weight-based separations of fully denatured and solubilized proteins in two dimensions. This doubled SDS-PAGE (dSDS-PAGE) approach produced a diagonal arrangement of protein spots and successfully circumvented problems associated with membrane proteome analysis involving traditional gel-based methods. Membrane proteins from the anaerobic bacterium Clostridium thermocellum were used for these investigations. Tricine-dSDS-PAGE and the newly developed Bicine-dSDS-PAGE were compared with the standard glycine-dSDS-PAGE (Laemmli protocol) in their suitability to separate C. thermocellum membrane proteins. Large-format gel experiments using optimized gel preparation and running buffer conditions revealed a 112% increase in protein spot count for Tricine-dSDS-PAGE and a 151% increase for Bicine-dSDS-PAGE, compared to glycine-dSDS-PAGE. The data clearly indicated that Bicine-dSDS-PAGE is a superior method for the analysis of membrane proteins, providing enhanced resolution and protein representation.
Williams
Taufika Islam
TI
Department of Chemistry, University of Kentucky, Lexington, KY 40506-0286, USA.
Combs
Jennifer C
JC
Thakur
Anup P
AP
Strobel
Herbert J
HJ
Lynn
Bert C
BC
D001426:Bacterial Proteins / Q000032:analysis / Q000302:isolation & purification; D002021:Buffers; D048013:Acetivibrio thermocellus / Q000737:chemistry; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification
D001426:Bacterial Proteins; D002021:Buffers; D008565:Membrane Proteins; C027494:N,N-bis(2-hydroxyethyl)glycine; D005998:Glycine