The changes of environmental oxygen levels induce a number of metabolic changes with rapid and profound consequences for cell physiology. To regulate oxygen homeostasis, the transcription factor hypoxia-inducible factor (HIF) regulates the expression of up to 50 adaptive genes by binding to hypoxia response elements (HRE) in their promoters. HIF consists of two subunits: oxygen-regulated α-subunit (HIF-1α) and constitutively expressed nuclear β-subunit (HIF-1β). In normoxia, HIF-1α is hydroxylated by oxygen-sensitive prolyl hydroxylases (PHDs) at Pro-402 and/or Pro-564 and asparaginyl hydroxylase FIH at Asn-80. Modified HIF-1α is subsequently destined to be degrade or prevented from the recruitment of the transcriptional coactivators p300 and CBP, resulting in low protein levels within cellular nuclear. Due to high availability of oxygen in hypoxia, however, hydroxylase activity decreases, thus enabling HIF-1a to accumulate and translocate to the nucleus where it forms transcriptional complex with the HIF1b/ARNT subunit and binds to HRE sequence to initiate gene expression.

Upon receipt, the positive control should be removed and stored at -20^° or -80^°C. The remainder of the kit can be stored for up to 6 months at 2-8^°C from the date of shipment. Opened Microplate Wells or reagents may be stored for up to 1 month at 2^° to 8^°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge. Note: The kit can be used within one year if the whole kit is stored at -20^°C upon receipt. Avoid repeated freeze-thaw cycles.