Bradford bioassay bradford protein assay kit

Tissue and whole-cell lysates were prepared on ice in radio immunoprecipitation assay supplemented with protease inhibitors, including PMSF, Na₃VO₄ and NaF, plus cocktail protein inhibitor. The concentration of protein samples was determined by the Bradford bioassay-Bradford protein assay kit (Sangon). Protein samples were electrophoresed in suitable SDS-PAGE gels and transferred in low temperature to polyvinylidene difluoride membranes (Amersham Bioscience). Fat-free milk (5%) was used to block blots at room temperature for 1 h, and then the blots were incubated with primary antibody overnight at 4 °C. After being washed with tris-buffered saline and 0.5% Tween-20, the blots were incubated with corresponding secondary antibody for 1 h at room temperature. Immunoreactive bands were visualized by chemiluminescence.

The total cell lysate was collected on ice using lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1 mM EDTA, 1 mM dithiothreitol and protease inhibitors, including phenylmethylsulfonyl fluoride, Na3VO4 and NaF, plus cocktail protein inhibitor). The concentration of protein samples was determined by the Bradford bioassay—Bradford protein assay kit (Sangon, Shanghai, China). Protein samples were electrophoresed in suitable SDS-PAGE gels and transferred in low temperature to PVDF membranes (Amersham Bioscience, Amersham, UK). Fat-free milk (5%) was used to block blots at room temperature for 1 h, then the blots were incubated with primary antibody overnight at 4 °C. After being washed with Tris-buffered saline and Tween 20 (TBST), the blots were incubated with corresponding secondary antibody for 1 h at room temperature. Proteins were visualized using enhanced chemiluminescence substrate (Tanon, Shanghai, China) and then quantified using a Tanon Chemiluminescent Imaging System.