Active Hypoxia Inducible Factor 1 Alpha (HIF1a)

HIF1a (Hypoxia-inducible factor 1-alpha) is a subunit of a transcription factor, which functions as a master transcriptional regulator of the adaptive response to hypoxia. The protein chaperone heat shock protein 90 (Hsp90) is a major regulator of different transcription factors, including HIF1a, thus a binding ELISA assay was conducted to detect the interaction of HIF1a and HSP90. Briefly, recombinant human HIF1a were diluted serially in PBS, with 0.01%BSA (pH 7.4). Duplicate samples of 100uL were then transferred to HSP90-coated microtiter wells and incubated for 2h at 37°C. Wells were washed with PBST and incubated for 1h with anti-HIF1a pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37°C. Finally, add 50µL stop solution to the wells and read at 450nm immediately. The binding activity of of HIF1a and HSP90 was shown in Figure 1, and this effect was in a dose dependent manner.