HIF-1 Alpha DNA Binding Activity (HIF) Assay Kit

Principle of the Assay: The HIF-1 Alpha DNA Binding Activity Assay Kit is an ELISA for the measurement of activated HIF-1. A biotinylated double stranded DNA containing an HRE is first bound to the provided Streptavidin plate wells. Then protein samples are added to the DNA conjugated microplate. After a brief incubation, an anti-HIF-1 Alpha antibody is added, followed by an HRP conjugated secondary antibody, allowing for simple colorimetric detection by plate spectophotometry.

Background: Mammalian cells are able to sense low oxygen conditions and turn on a series of genes in response to the lack of oxygen. The hypoxia-inducible factor 1 transcriptional activator complex (HIF-1) is involved in the activation of several hypoxia-responsive genes including erythropoietin and VEGF. The HIF-1 complex is composed of two protein subunits: the constitutively expressed HIF-1b/ARNT (aryl hydrocarbon receptor nuclear translocator), and HIF-1 Alpha, the latter of which is not detected during normoxia since it is continually degraded by the ubiquitin proteasome system (UPS). In the presence of low oxygen conditions, however, HIF-1 Alpha is stabilized, accumulates, translocates from the cytosol to the nucleus, dimerizes with HIF-1b/ARNT, and becomes transcriptionally active. Activated HIF-1 complex then associates with hypoxic response elements (HREs) and binds transcriptional coactivators to induce gene expression. Tight regulation of the stability and function of HIF-1 is controlled by its post-translational modifications, such as hydroxylation, ubiquitination, acetylation, and phosphorylation. Under normal oxygen conditions, the post-translational modification of HIF-1 Alpha occurs within several domains: hydroxylation of two proline residues and acetylation of a lysine residue in its oxygen dependent degradation domain (ODDD) promote binding of HIF-1 with the von Hippel-Lindau (pVHL) ubiquitin E3 ligase complex. This pVHL complex modifies HIF-1 with ubiquitin, marking it for degradation by the 26S proteasome. Furthermore, hydroxylation of C-terminal asparagine residue in the c-terminal transactivation domain blocks association of HIF-1 with CBP/p300 and as a result inhibits HIF-1 transcriptional activity. Upon synthesis of HIF-1 Alpha, the protein is rapidly hydroxylated by a family of 2-oxoglutarate dioxygenases on proline 402 and 564. Hypoxic or chemical inactivation of these dioxygenases (which were later termed proline hydroxylase domains (PHDs)), leads to an increase in the half life of HIF-1 Alpha and subsequent activation of HIF-1 complex. HIF-1 Alpha DNA Binding Activity Assay Kit is an immunoassay developed for rapid detection of activated HIF-1 in any protein sample. Active HIF-1 complex is captured on a double stranded oligonucleotide containing an HRE that is attached to the well. HIF-1 Alpha is then detected with an anti-HIF-1 Alpha antibody followed by an HRP conjugated secondary antibody. Each kit provides sufficient reagents to perform up to a total of 96 assays, and can detect HIF-1 Alpha from human, mouse, or rat. The HIF-1 DNA Binding Activity Assay takes only 4.5 hours to complete compared to a standard 3 day electromobility shift (EMSA) method.

1. Ke Q and Costa M. (2006) Molecular Pharm. 70:1469-1480.
2. Salceda S and Caro J (1997) J. Biol. Chem. 272: 22642-22647.
3. CW Pugh, JF O'Rourke, M Nagao, JM Gleadle, and PJ Ratcliffe (1997) J. Biol. Chem. 272: 11205-11214.
4. LE Huang, Z Arany, DM Livingston, and HF Bunn (1996) J. Biol. Chem. 272: 32253-32259.
5. PJ Kallio, I Pongratz, K Gradin, J McGuire, and L Poellinger (1997) Proc Natl Acad Sci USA 94: 5667-5672

Small volumes of HIF-1 Alpha DNA Binding Activity assay kit vial(s) may occasionally become entrapped in the seal of the product vial during shipment and storage. If necessary, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container`s cap. Certain products may require to ship with dry ice and additional dry ice fee may apply.

MBS168165 is an assay kit targeting HIF-1 Alpha DNA Binding Activity.