Nori Human HIF-1A ELISA Kit Summary

Alternative names for HIF-1A: Hypoxia-inducible factor 1-alpha

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background:

Hypoxia-inducible factor 1-alpha (HIF-1-alpha) is a subunit of a heterodimeric transcription factor HIF-1 that is encoded by the HIF1A gene.^[1] It is a basic helix-loop-helix PAS domain containing protein, and is considered as the master transcriptional regulator of cellular and developmental response to hypoxia.^[2] HIF1A expression level is dependent on its GC-rich promoter activation.^[3] In most cells, HIF1A gene is constitutively expressed in low levels under normoxic conditions, however, under hypoxia, HIF1A transcription is often significantly upregulated.^[3] Typically, oxygen-independent pathway regulates protein expression, and oxygen-dependent pathway regulates degradation.^[4] In hypoxia-independent ways, HIF1A expression may be upregulated through a redox-sensitive mechanism.^[5]  HIF-1 plays an important role in cellular response to systemic oxygen levels in mammals. HIF1A activity is regulated by a host of post-translational modifications: hydroxylation, acetylation, and phosphorylation. HIF-1 is known to induce transcription of more than 60 genes, including VEGF and erythropoietin, which assist in promoting and increasing oxygen delivery to hypoxic regions.^[4] HIF-1 also induces transcription of genes involved in cell proliferation and survival, as well as glucose and iron metabolism. HIF-1 responds to systemic oxygen levels by undergoing conformational changes, and associates with HRE regions of promoters of hypoxia-responsive genes to induce transcription.^[6] HIF1A stability, subcellular localization, as well as transcriptional activity are especially affected by oxygen level. Under normoxic conditions, VHL-mediated ubiquitin protease pathway rapidly degrades HIF1a; however, under hypoxia, HIF1A protein degradation is prevented and HIF1A levels accumulate to associate with HIF1B to exert transcriptional roles on target genes ^[7] Following axon injury, HIF1A activates VEGFA to promote regeneration and functional recovery.^[8] HIF1A abundance is regulated transcriptionally in an NF-κB-dependent manner.^[9] HIF-1 is overexpressed in many human cancers.^[10] HIF-1 overexpression is heavily implicated in promoting tumor growth and metastasis through its role in initiating angiogenesis and regulating cellular metabolism to overcome hypoxia.^[11] Hypoxia promotes apoptosis in both normal and tumor cells.

References

1. Semenza GL, et al. (1996) Genomics. 34 (3): 437–9.
2. Wang GL, et al. (1995). Proc Natl Acad Sci USA. 92 (12): 5510–4.
3. Minet E, et al. (1999). Biochemical and Biophysical Research Communications. 261 (2): 534–40.
4. Semenza GL (2003). Nature Reviews. Cancer. 3 (10): 721–32.
5. Bonello S, et al. (2007). Arteriosclerosis, Thrombosis, and Vascular Biology. 27 (4): 755–61.
6. Epstein AC, et al. (2001). Cell. 107 (1): 43–54.
7. Kallio PJ, et al. (1997). Proc Natl Acad Sci USA 94 (11): 5667–72.
8. Cho Y, et al. (2015). Neuron. 88 (4): 720–34.
9. van Uden P, et al. (2008). The Biochemical Journal. 412 (3): 477–84.
10. Zhong H, et al. (1999). Cancer Research. 59 (22): 5830–5.
11. Bos R, et al. (2003). Cancer. 97 (6): 1573–81.