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

Product Summary

This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure HIF-2 alpha / EPAS1 in cell lysates. An immobilized capture antibody specific for HIF-2 alpha / EPAS1 binds both phosphorylated and unphosphorylated HIF-2 alpha / EPAS1. After washing away unbound material, a biotinylated detection antibody is used to detect both phosphorylated and unphosphorylated protein, utilizing a standard Streptavidin-HRP format.

Preparation and Storage

Background: HIF-2 alpha/EPAS1

The hypoxia-inducible transcription factor 2 alpha is stabilized in hypoxic tissue and, similarly to HIF-1 alpha, complexes with aryl hydrocarbon receptor nuclear translocator (ARNT). Both the HIF-1 and HIF-2 complexes bind hypoxia-response elements (HREs) in the promoters of many genes involved in adapting to an environment of insufficient oxygen or hypoxia. HIF-1 and HIF-2 do not appear to be completely redundant, although specific functions are only beginning to be elucidated. Hypoxic tissue environments occur in vascular and pulmonary diseases as well as cancer, indicating the potentially broad impact of gene regulation by both HIF-1 alpha and HIF-2 alpha.

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1、Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2、Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3、Add 50 μL of Assay Diluent to each well.

4、Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5、Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6、Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7、Aspirate and wash 5 times.

8、Add 100 μL Substrate Solution to each well.

9、Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.