Human apoptosis antibody array kit

A human apoptosis antibody array kit (Ray Biotech, Norcross, GA) was used to detect the relative levels of apoptosis-related proteins in the cell lysates according to the manufacturer's instructions. Briefly, the treated cell lysates were added to the antibody array membranes and incubated overnight with rocking at 4°C. After washing membrane, the membranes were incubated with a cocktail of biotin-conjugated anti-antibodies to apoptotic proteins at room temperature for 2 hours. After incubation with horseradish peroxidase–streptavidin at room temperature for 1.5 hours, the signals were visualized by chemiluminescence.

The Human Apoptosis Antibody Array Kit (RayBiotech, USA) was used to examine multiple apoptotic regulators according to the manufacturer's instructions. Briefly, 1 × 10⁵ cells were plated in each well of a 12-well plate and treated with 50 μM DMDD for 6 h. The cells were then lysed with Cell Lysis Buffer containing proteinase inhibitor from the kit. The cell lysate was concentrated using a protein concentration spin column (EMD Millipore, USA) to a 2 mg/ml concentration. After blocking, the cell lysate was incubated with the glass chip provided in the kit for 2 h at room temperature. Following five washes, the cocktail of biotin-conjugated antibodies was added to the glass chip and incubated overnight at 4°C. Afterwards, HiLyte Plus-conjugated streptavidin was added to the chip and incubated for 2 h at room temperature. Image scanning and data extraction was completed by RayBiotech. Fluorescent intensities from each dot of the array were normalized to the internal control.