Anti his hrp conjugate antibody

Cell pellets were resuspended according to their optical density (100 μl per OD₆₀₀ of 1) in 1 x SDS sample buffer (2% SDS, 0.1% bromophenol blue, 1% 2-mercaptoethanol, 25% glycerol, 50 mM Tris/HCl, pH 6.8). The samples were boiled for 10 min at 95°C and after centrifugation (10 min, 13000 rpm), the supernatant was loaded and separated by SDS gel electrophoresis in 5% stacking and 12% separating gels. By tank blotting, the proteins were transferred onto a nitrocellulose membrane (Hybond-C Extra, GE Healthcare) and an anti-His-HRP conjugate antibody (Bio-Rad) was used in a 1:4000 dilution. Chemiluminescence signals were detected by incubating membranes with Immobilon Forte Western HRP substrate (Millipore) with a ChemiDoc MP Imaging System (Biorad).

Cell pellets were resuspended in 1 × SDS sample buffer (2% SDS, 0.1% bromophenol blue, 1% 2-mercaptoethanol, 25% glycerol, 50 mM Tris/HCl, and pH 6.8) according to their optical density (100 μl per OD₆₀₀ of 1). After boiling for 10 min at 95°C, samples were centrifugated (10 min, 13,000 rpm) and the supernatant was separated by SDS gel electrophoresis in 5% stacking and 12% separating gels. Size-separated proteins were transferred by tank blotting onto a nitrocellulose membrane (Hybond-C Extra, GE Healthcare). An anti-His-HRP conjugate antibody (Bio-Rad) was used in a 1:4,000 dilution. Luminescence signals were detected by incubating membranes with Immobilon Forte Western HRP substrate (Millipore) and the FluorChem SP (Alpha Innotec).