HIF-1 alpha [Hydroxy Pro564] Antibody - BSA Free

Western Blot: HIF-1 alpha [Hydroxy Pro564] Antibody [NB110-74679] - Detection (left panel) of HIF-1 alpha [Hydroxy Pro564] in nuclear extracts of A549 cells treated with MG132 (a proteasome inhibitor). Hydroxyproline is not recognized on HIF-1alpha when cells are first treated with DFO, a prolyl hydroxylase inhibitor that prevents HIF hydroxylation. Control staining is shown (right panel) using conventional anti-HIF-1alpha. The asterisk marks a band approximately 110 kDa molecular weight in size corresponding to HIF-1 alpha. The primary antibody was used at a 1:1000 dilution in 2% BLOTTO.

• Reactivity: Hu, Mu
• Applications: WB, ELISA, IHC, WB
• Clonality: Polyclonal
• Host: Rabbit
• Conjugate: Unconjugated
• Format: BSA Free

HIF-1 alpha [Hydroxy Pro564] Antibody - BSA Free Summary

• Description: This antibody is directed against human HIF-1a hydroxy P564 and is specific for the hydroxylated form of the protein
  
  Store vial at -20C prior to opening. Aliquot contents and freeze at -20C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4C as an undiluted liquid. Dilute only prior to immediate use.
• Immunogen: This HIF-1 alpha [Hydroxy Pro564] Antibody was prepared from whole rabbit serum produced by repeated immunizations with a synthetic peptide corresponding to a region surrounding the P564 of human HIF-1 alpha. (Uniprot: Q16665)
• Modification: Hydroxy Pro564
• Localization: Cytoplasmic in normoxia, nuclear translocation in response to hypoxia.
• Specificity: This antibody is directed against human HIF-1a hydroxyP564 and is specific for the hydroxylated form of the protein. Minimal reactivity occurs with the non-hydroxylated form of the protein. This antibody is specific for HIF-1a hydroxylated at P564. Minimal cross-reactivity occurs with non-hydroxylated HIF-1a. A BLAST analysis was used to suggest cross-reactivity with HIF-1a from human, monkey, mouse, rat, dog, bovine and Xenopus sources based on a 100% homology with the immunizing sequence. Reactivity against homologues from other sources is not known.
• Isotype: Serum
• Clonality: Polyclonal
• Host: Rabbit
• Gene: HIF1A
• Purity: Unpurified

Applications/Dilutions

• Dilutions:
  • ELISA 1:5000-1:20000
  • Immunoblotting 1:100-1:2000
  • Immunohistochemistry 1:10 - 1:500
  • Immunohistochemistry-Paraffin 1:10 - 1:500
  • Western Blot 1:500-1:2000
• Application Notes: This antibody has been tested for use in ELISA and western blotting. Specific conditions for reactivity should be optimized by the end user. Expect a band approximately 110 kDa in size corresponding to HIF-1a hydroxyl P564 by western blotting in the appropriate cell lysate or extract.
  
  Use in Immunoblotting reported in scientific literature (PMID 28566998).
• Control:

  Human Pancreas Whole Tissue Lysate (Adult Whole Normal)
  HIF-1 alpha Knockout CoCl2-treated/untreated HeLa Cell Lysate
  HIF-1 alpha Knockout Hypoxic-treated/untreated HeLa Cell Lysate
  NIH 3T3 CoCl2 treated/Untreated Cell Lysate
  L929 CoCl2 treated/Untreated Cell Lysate
  Raw 264.7 CoCl2 treated/Untreated Cell Lysate
  Raw 264.7 Hypoxic / Normoxic Cell Lysate
  L929 Hypoxic / Normoxic Cell Lysate
  NIH 3T3 Hypoxic / Normoxic Cell Lysate

Reactivity Notes

A BLAST analysis was used to suggest cross-reactivity with HIF-1 alpha from monkey, rat, dog, bovine and Xenopus sources based on a 100% homology with the immunizing sequence. Reactivity against homologues from other sources is not known

Packaging, Storage & Formulations

• Storage: Store at -20C short term. Aliquot and store at -80C long term. Avoid freeze-thaw cycles.
• Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
• Preservative: 0.01% Sodium Azide
• Purity: Unpurified

Alternate Names for HIF-1 alpha [Hydroxy Pro564] Antibody - BSA Free

• AINT
• anti-HIF-1 alpha
• anti-HIF1A
• ARNT interacting protein
• ARNT-interacting protein
• Basic-helix-loop-helix-PAS protein MOP1
• BHLHE78
• Class E basic helix-loop-helix protein 78
• HIF 1A
• HIF1 alpha
• HIF-1 alpha
• HIF1
• HIF1A
• HIF-1a
• HIF-1alpha
• HIF-1-alpha
• HIF1-alpha
• hypoxia inducible factor 1 alpha subunit, hypoxia inducible factor 1 subunit alpha
• hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)
• hypoxia-inducible factor 1-alpha
• Member of PAS protein 1
• member of PAS superfamily 1
• MOP1
• PAS domain-containing protein 8
• PASD8

Background

Hypoxia contributes to the pathophysiology of human disease, including myocardial and cerebral ischemia, cancer, pulmonary hypertension, congenital heart disease and chronic obstructive pulmonary disease (1). In cancer and particularly solid tumors, hypoxia plays a critical role in the regulation of genes involved in stem cell renewal, epithelial to mesenchymal transition (EMT), metastasis and angiogenesis. In the tumor microenvironment (TME), hypoxia influences the properties and function of stromal cells (e.g., fibroblasts, endothelial and immune cells) and is a strong determinant of tumor progression (2,3).

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

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for HIF-1 alpha Antibody (NB110-74679)(7)

Publications using NB110-74679

• Badawi Y, Shi H. Relative Contribution of Prolyl Hydroxylase-Dependent and -Independent Degradation of HIF-1alpha by Proteasomal Pathways in Cerebral Ischemia. Front Neurosci 2017-06-01 [PMID: 28566998] (IB, Mouse)
• Ohgami N, Yajima I, Iida M et al. Manganese-mediated acceleration of age-related hearing loss in mice. Sci Rep. 2016-11-08 [PMID: 27824154] (IF/IHC, Mouse)
• Malik RA, Zippel N, Fromel T et al. AMP-Activated Protein Kinase alpha2 in Neutrophils Regulates Vascular Repair via Hypoxia-Inducible Factor-1alpha and a Network of Proteins Affecting Metabolism and Apoptosis. Circulation Research. 2016-10-24 [PMID: 27777247] (WB, Mouse)
• Yeh IJ, Ogba N, Bensigner H et al. HEXIM1 downregulates Hypoxia Inducible Factor-1 alpha protein stability. Biochem J. 2013-09-10 [PMID: 24015760] (WB, Human)
• Matoba K, Kawanami D, Okada R et al. Rho-kinase inhibition prevents the progression of diabetic nephropathy by downregulating hypoxia-inducible factor 1alpha. Kidney Int 2013-04-24 [PMID: 23615507] (WB, Mouse)
• Roitbak T, Surviladze Z, Cunningham LA. Continuous expression of HIF-1alpha in neural stem/progenitor cells Cell Mol Neurobiol 2011-01-01 [PMID: 20844947] (WB, Mouse)
• Yeh IJ. HEXIM1 IS AN INHIBITOR OF TWO TRANSCRIPTION FACTORS CRITICAL IN CANCER: THE ANDROGEN RECEPTOR AND HYPOXIA INDUCIBLE FACTOR-1 ALPHA Thesis. 2015-01-01 (WB, Human)

Reviews for HIF-1 alpha Antibody (NB110-74679) (3)

Average Rating: 4

(Based on 3 reviews)

We have 3 reviews tested in 2 species: Human, Other.

reviewed by: Verified Customer WB Human 09/02/2015

Summary

• Application: Western Blot
• Sample Tested: Human breast cancer cell lines
• Species: Human

reviewed by: Verified Customer WB Other 06/02/2014

Summary

• Application: Western Blot
• Sample Tested: PC12 cell whole cell lysate.
• Species: Other
• Lot: 19691

reviewed by: Verified Customer WB Other 03/28/2014

Summary

• Application: Western Blot
• Sample Tested: PC12 whole cell lysate
• Species: Other
• Lot: 19691

FAQs for HIF-1 alpha Antibody (NB110-74679). (Showing 1 - 10 of 11 FAQs).

1. Why is there a difference between the theoretical MW for HIF1A and the observed MW for HIF-1 alpha?
   • HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.
2. Which antibody(ies) do you recommend for the detection of HIF-1a by immunohistochemistry in the sections of paraffin-embedded mouse liver samples? I would appreciate if you can give me several choices and rank them in the order of performance. My goal is to distinguish HIF upregulation by prolyl hydroxylase inhibitor in different liver cells.
   • All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).
3. I would like to know, does a path exist for detection of HIF 1 in venous blood before and after revascularization of the leg? 
   • We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.
4. What is the molecular weight (kDa) of protein HIF 1 alpha in western blot?
   • The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.
5. We got the Hif1a (NB100-105) antibody from you guys. I used the concentration that is mentioned on your website, but I am getting a band of a completely different size (~70kDa) and not the 120 kDa mentioned.
   • HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.Please go through our hypoxia related FAQs, you should find them very informative.Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)
6. I performed several Western Blots of HIF-1 alpha with different lysis buffers, whole lysates, and cytoplasm/nuclei extractions. I can’t seem to get a good western blot (poor signal, band much lower than expected, etc.). Can someone suggest some technical considerations/tricks I should consider using?
   • A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.
7. I am doing HIF1 westerns in HIF-overexpressing mouse liver and adipose tissue using Novus antirabbit HIF1a antibody with overnight incubation. I am getting strong bands around 90kDa. I am aware that HIF theoretical molecular weight is 93kDa, but in westerns, the HIF band is usually around 120kDa according to my internet research. Can someone let me know if I’m getting the right HIF band or just some non-specific bands? Thanks.
   • (1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs (5)    Last but not the least, Novus technical support team may be contacted via email
8. I have Hif1a nuclear protein extract at -80C. I am wondering if anyone knows how long it would be good for at that temperature since HIf1a is known to be degraded easily.Thank you!
   • You could try a few things to further inhibit the degradation.1) Use the protease inhibitors (if you are not already using them).2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.
9. I am curious to know the biochemical reactions of CoCl2 that mimic hypoxia. Is it that CoCl2 can bind any ubiquitin enzyme which regulates their degradation?
   • CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.
10. I am curious to know the biochemical reactions of CoCl2 that mimic hypoxia. Is it that CoCl2 can bind any ubiquitin enzyme which regulates their degradation?
    • CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.
11. Is cross-reactivity with HIF-2 alpha tested/predicted?
    • Although we don’t have cross-reactivity data with regards to HIF-2 alpha, we predict minimal cross-reactivity based on low sequence similarity observed from BLAST analysis between HIF-1 alpha and HIF-2 alpha.
Show All 11 FAQs.

Bioinformatics

• Gene Symbol: HIF1A
• Entrez:
  • 3091(Human)
• Uniprot:
  • Q16665()
  • Q16665()
  • Q16665()
  • Q16665()