HIF-1 alpha Knockout Hypoxic-treated/untreated HeLa Cell Lysate
Novus Biologicals, part of Bio-Techne | Catalog # NBP3-11827
Key Product Details
Species
Human
Applications
SDS-PAGE
Product Specifications
Applications
SDS-Page (10 ul / lane)
Application Notes
HIF-1 alpha KO HeLa Whole cell lysate is provided as a Western blot positive control for Hif-1 research. The lysates are provided as a set of hypoxic and normoxic samples. Use 10 ul per lane for a standard mini-gel blot (approx. 1 mg/ml).
Type
Knockout Hypoxic-treated/untreated HeLa Cell
Subcellular Fraction
Hypoxic / Normoxic
Kit Contents for HIF-1 alpha Knockout Hypoxic-treated/untreated HeLa Cell Lysate
- 0.1 ml HIF-1 alpha Knockout Hela Hypoxic whole cell lysate
- 0.1 ml HIF-1 alpha Knockout Hela Normoxic whole cell lysate
Formulation, Preparation, and Storage
Preparation Method
HIF-1 alpha KO HeLa cells are grown under standard tissue culture conditions until semi-confluent (70-80%). The cells are grown under true hypoxic conditions where the oxygen level in the incubator is reduced to 2% for 4 hours to accumulate Hif-1a within the cells. The cells are then washed in PBS and directly lysed into 1X Laemmli sample buffer with BME. The lysate is sonicated and tested by Western blot.
Formulation
The protein lysates are prepared in 1x Laemmli sample buffer with BME. Boiling before loading is not necessary.
Concentration
1.0 mg/ml
Storage
Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.
Background: HIF-1 alpha/HIF1A
HIF-1 or hypoxia inducible factor 1 (predicted molecular weight 93kDa), is a transcription factor commonly referred to as a "master regulator of the hypoxic response" for its central role in the regulation of cellular adaptations to hypoxia. In its active form under hypoxic conditions, HIF-1 is stabilized by the formation of a heterodimer of HIF-1 alpha and ARNT/HIF-1 beta subunits. Nuclear HIF-1 engages p300/CBP for binding to hypoxic response elements (HREs). This process induces transcription and regulation of genes including EPO, VEGF, iNOS2, ANGPT1 and OCT4 (4,5).
Under normoxic conditions, the HIF-1 alpha subunit is rapidly targeted and degraded by the ubiquitin proteasome system. This process is mediated by prolyl hydroxylase domain enzymes (PHDs), which catalyze the hydroxylation of key proline residues (Pro-402 and Pro-564) within the oxygen-dependent degradation domain of HIF-1 alpha. Once hydroxylated, HIF-1 alpha binds the von Hippel-Lindau tumor suppressor protein (pVHL) for subsequent ubiquitination and proteasomal degradation (4). pVHL dependent regulation of HIF-1 alpha plays a role in normal physiology and disease states. Regulation of HIF-1 alpha by pVHL is critical for the suppressive function of FoxP3+ regulatory Tcells (6). Repression of pVHL expression in chronic lymphocytic leukemia (CLL) B cells leads to HIF-1 alpha stabilization and increased VEGF secretion (7).
References
1. Semenza, G. L., Agani, F., Feldser, D., Iyer, N., Kotch, L., Laughner, E., & Yu, A. (2000). Hypoxia, HIF-1, and the pathophysiology of common human diseases. Advances in Experimental Medicine and Biology.
2. Muz, B., de la Puente, P., Azab, F., & Azab, A. K. (2015). The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy. Hypoxia. https://doi.org/10.2147/hp.s93413
3. Huang, Y., Lin, D., & Taniguchi, C. M. (2017). Hypoxia inducible factor (HIF) in the tumor microenvironment: friend or foe? Science China Life Sciences. https://doi.org/10.1007/s11427-017-9178-y
4. Koyasu, S., Kobayashi, M., Goto, Y., Hiraoka, M., & Harada, H. (2018). Regulatory mechanisms of hypoxia-inducible factor 1 activity: Two decades of knowledge. Cancer Science. https://doi.org/10.1111/cas.13483
5. Dengler, V. L., Galbraith, M. D., & Espinosa, J. M. (2014). Transcriptional regulation by hypoxia inducible factors. Critical Reviews in Biochemistry and Molecular Biology. https://doi.org/10.3109/10409238.2013.838205
6. Lee, J. H., Elly, C., Park, Y., & Liu, Y. C. (2015). E3Ubiquitin Ligase VHL Regulates Hypoxia-Inducible Factor-1 alpha to Maintain Regulatory T Cell Stability and Suppressive Capacity. Immunity. https://doi.org/10.1016/j.immuni.2015.05.016
7. Ghosh, A. K., Shanafelt, T. D., Cimmino, A., Taccioli, C., Volinia, S., Liu, C. G., ... Kay, N. E. (2009). Aberrant regulation of pVHL levels by microRNA promotes the HIF/VEGF axis in CLL B cells. Blood. https://doi.org/10.1182/blood-2008-10-185686
Long Name
Hypoxia Inducible Factor 1 Subunit Alpha
Alternate Names
BHLHE78, HIF 1A, HIF-1a, HIF1 alpha, HIF1A, MOP1, PASD8
Gene Symbol
HIF1A
Additional HIF-1 alpha/HIF1A Products
Product Documents for HIF-1 alpha Knockout Hypoxic-treated/untreated HeLa Cell Lysate
Product Specific Notices for HIF-1 alpha Knockout Hypoxic-treated/untreated HeLa Cell Lysate
This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.
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