Proteome profiler antibody array kit

Protein profile array was done to determine expression of some apoptotic proteins of the extrinsic pathway in clausenidin-treated HepG2 cells using the proteome profiler antibody array kit (R and D systems, USA). Briefly, cells were seeded overnight at a density of 1 × 10⁶ cells/well and treated with 15 μg/mL clausenidin while vehicle control cells treated with 0.1% (v/v) DMSO, both for 24 h. The protein concentrations were determined by Bradford’s assay. Primary antibody-coated membranes (R and D systems, USA) were incubated with protein samples from HepG2 cells at a concentration range of 200 to 400 μg/mL overnight at 4°C and washed with wash buffer before incubating with secondary antibody conjugated with streptavidin-bound horseradish peroxidase (R and D systems, USA) for 30 min on a rocking platform. The membranes were again washed and chemireagent mix was slowly pipetted onto the membrane to ensure that the membrane surface is fully covered. The images were then captured on a ChemiDocTM Imaging system (Biorad, USA) and the data obtained was analyzed using the Image Lab software 5.