Ppar gamma transcription factor assay kit

PPARγ activity was measured using PPAR gamma Transcription Factor Assay Kit (ab133101, Abcam) following the manufacturer’s instructions. Briefly, nuclear extracts were isolated from cultured HBMECs using “Nuclear Extraction Kit” (ab113474, Abcam). Ten microliters of nuclear extract from HBMECs or positive control was mixed with 90 µL Complete Transcription Factor Binding Assay Buffer (CTFB) and added to designated wells of a 96-well assay plate. The plate was then sealed and incubated at 4 °C overnight. The next day, the wells were emptied and washed five times with 200 µL of Wash Buffer. Transcription Factor PPAR gamma Primary Antibody (100 µL, 1:100) was then added to each well and incubated at room temperature for 1 h without agitation. After five times wash, goat anti-Rabbit HRP conjugate (1:100) was added to each well, followed by 1 h incubation at room temperature. After washing, a 100 µL developing solution was added and incubated at room temperature for 15 to 45 min, followed by adding a 100 µL stop solution. Absorbance at 450 nm was measured using a plate reader.