Goat anti mouse igg hrp

Cell lysates were prepared by sonication with retic buffer followed by further sonication with the addition of urea buffer (7M urea, 2.8M thiourea, 4% CHAPS, 130 mM dithiothreitol, and 40mM Tris-HCl (pH 8.8)) at a 1:1 ratio. After centrifugation, the supernatant was collected, and protein quantification was performed using the Bradford assay (Bio-rad, USA). Equivalent amounts of proteins (10 μg per lane) were loaded and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Afterward, proteins were transferred to a nitrocellulose membrane. The membrane was blocked for 1 hr using 5% skim milk (BD Difco, USA) in Tris-buffered saline with 0.1% Tween 20(TBST) and then incubated with the anti-ubiquitin antibody (Ub, 1:1000; Santa Cruz, USA) at 4 °C overnight. Anti-beta(β)-actin antibody (1:5000; Santa Cruz, USA) was used as a loading control. Afterward, the membrane was incubated with the secondary antibody (goat anti-mouse IgG-HRP; Ab Frontier, USA) for 1 hr at RT. Blots were developed using ECL (Advansta, USA), and protein bands were detected through exposure to X-ray film. Densitometric analysis was performed using Image-J software (NIH, USA).