Sds page molecular weight standards

Chromatographic resins Phenyl Sepharose, Butyl Sepharose and CNBr-activated Sepharose 4B were from GE Lifesciences (Marlborough, MA, USA); Butyl Toyopearl was from Tosoh Bioscience (Griesheim, Germany). The SDS-PAGE molecular weight standards and bicinchoninic acid (BCA) protein assay kit were from Thermo Fisher Scientific (Rockford, IL, USA). Other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), Merck (Darmstadt, Germany), Fluka (Buchs, Switzerland), PanReac AppliChem ITW Reagents (Darmstadt, Germany), Serva (Heidelberg, Germany) and Amresco (Solon, OH, USA) and were at least analytical grade. All buffers and other solutions were prepared using ultrapure water.

Prior to SDS-PAGE, the gel strips were incubated in equilibration buffer I (50 mM Tris-HCl, pH 8.8, 6 M urea, 2% SDS, 30% glycerol and 1% DTT) for 15 min and then in equilibration buffer II (50 mM Tris-HCl, pH 8.8, 6 M urea, 2% SDS, 30% glycerol and 2.5% iodoacetamide) for 15 min. The strips and SDS-PAGE molecular weight standards (11–180 kDa; Thermo Fisher Scientific, Waltham, MA, USA) were then loaded onto the 12.5% polyacrylamide gels [21 (link)]. Each sample was simultaneously run on 2 separate gels, one was used for western blotting, the other was used for silver staining, according to the methods reported previously [27 (link)]. ArtixScan 1010 Plus (Microtek International, Inc., Taiwan, China) was used for imaging gels.