Human/Rat Total HIF-2 alpha/EPAS1 DuoSet IC ELISA

Standard Curve

Total HIF-2 alpha Detection in HepG2 Cell Lysate.

Lysate prepared from HepG2 human hepatocellular carcinoma cells treated with 150 μM of CoCl2 for 18 hours were incubated in wells coated with Total HIF-2 alpha Capture Antibody. Unbound material was removed by washing and bound material was solubilized in SDS gel sample buffer. The same lysate and captured proteins were electrophoresed and transferred to a PVDF membrane. The captured protein was immunoblotted with Total HIF-2 alpha Detection Antibody and the lysate was immunoblotted with anti-HIF-2 alpha antibody. Only one band corresponding to HIF-2 alpha was detected in the captured material.Cross-reactivity experiments were performed with this DuoSet IC ELISA to further determine specificity. Recombinant mouse HIF-2 alpha was assayed at 80 ng/mL and read 16.4 ng/mL (20.5% cross-reactivity). Recombinant human (rh) HIF-1 alpha and rhARNT were assayed at 80 ng/mL and did not cross-react or interfere in the assay.

Total HIF-1 alpha Detection in 786-O, Caki-2, and MCF-7 Cell Lysate.

Lysate prepared from 786-O human renal adenocarcinoma cells, Caki-2 human kidney clear carcinoma cells, and MCF-7 human breast cancer cells untreated or treated with 150 μM of CoCl2 for 18 hours were analyzed with both this DuoSet IC ELISA and the Human/Mouse Total HIF-1 alpha DuoSet IC ELISA (R&D Systems, Catalog # DYC1935). The 786-O cells are positive for HIF-2 alpha only while Caki-2 and MCF-7 treated with CoCl2 are positive for HIF-1 alpha only. Only the HIF-2 alpha positive 786-O cells were detected with the Human/Rat Total HIF-2 alpha/EPAS1 DuoSet IC ELISA, indicating that this DuoSet IC ELISA is specific for HIF-2 alpha.