HIF-1 alpha Antibody Pack

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-901IHC] - Immunohistochemical analysis in non-GIST STS representing negative, and score 1-3 of Carbonic Anhydrase IX/CA9, GLUT-1, HIF-1 alpha, and HIF-2 alpha/EPAS1. non-GIST STS: non-gastrointestinal stromal tumor soft-tissue sarcomas, Carbonic Anhydrase IX/CA9: carbonic anhydrase IX, GLUT-1: glucose transporter-1, and HIF-1/2alpha: hypoxia induced factor 1/2alpha. Image collected and cropped by CiteAb from the following publication (//www.hindawi.com/journals/sarcoma/2012/541650/), licensed under a CC-BY licence. HIF-1 alpha Antibody (ESEE122) [NB100-131]

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-901IHC] - Immunohistochemical detection of HIF-1alpha protein in human tissues. The nature of the tissue is indicated on top of each figure. Original magnifications are as follows: A, x400; B, x400; C, x100; D, x100; E, x100; F, x100; G, x40; H, x40. Image collected and cropped by CiteAb from the following publication (//bmcgenet.biomedcentral.com/articles/10.1186/1471-2156-5-27), licensed under a CC-BY licence. HIF-1 alpha Antibody (H1alpha67) [NB100-123]

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-901IHC] - Staining in normal mouse kidney showing (A) staining with antibody and (B) blocking peptide used at 10-fold excess concentration. Images provided by Dr Yves Heremans. Please see the product review of NBC1-18422 for additional details. HIF-1 alpha Antibody [NB100-479]

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-901IHC] - Hif1alpha expression in preimplantation embryos. Co-staining of OCT4 and Hif1alpha in inner cell mass of blastocyst (top) and negative control images without primary antibodies (bottom). Scale bar: 30um. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0154309) licensed under a CC-BY licence. HIF-1 alpha Antibody [NB100-479]

Western Blot: HIF-1 alpha Antibody Pack [NB100-901IHC] - Normoxic pre-treatment sensitizes glioma cells to radiation after graded chronic hypoxic (GCH) exposure. The results of anchorage-independent colony forming assays are shown for U87, U87-luc, GL261 glioma cells and 0308 GSCs after 5 Gy radiation exposure under varying O2 conditions. Each result represents at least 3 independent samples, plated in triplicate. Holm-Sidak comparisons for multiple groups were used for statistical comparisons of raw values (*p<0.05, **p<0.01). Also shown are Western blots of nuclear HIF-1 alpha at the time of irradiation. Corresponding Western blots of lamin A/C are shown as a loading control. Image collected and cropped by CiteAb from the following publication (//dx.plos.org/10.1371/journal.pone.0111199) licensed under a CC-BY licence. HIF-1 alpha Antibody [NB100-479]

Immunocytochemistry/Immunofluorescence: HIF-1 alpha Antibody Pack [NB100-901IHC] - Detection of HIF-1 alpha (red dye) in a cell cytospin from a lavage of a murine skin pouch infected with S. aureus. 100X magnification. Blue: DAPI nuclear staining. HIF-1 alpha Antibody (ESEE122) [NB100-131]

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-901IHC] - Analysis of a FFPE mouse kidney tissue section using HIF-1 alpha antibody clone ESEE122 at 1ug/mL concentration. The detection was performed using X-cell plus universal HRP polymer detection system with Vector SG chromagen substrate. Image courtesy of a product review by Steven Grover. HIF-1 alpha Antibody (ESEE122) [NB100-131]

Western Blot: HIF-1 alpha Antibody Pack [NB100-901IHC] - HIF-1 alpha induction on Caki-1 cell lysate using CoCl2. Image from verified customer review. HIF-1 alpha Antibody [NB100-449]

Immunocytochemistry/Immunofluorescence: HIF-1 alpha Antibody Pack [NB100-901IHC] - Detection of HIF-1 alpha (red dye 568) in a cultured raw mouse macrophage cell line, using NB100-131. 100X magnification. Photos courtesy of Susan Alexander and Hattie Gresham, PhD.

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-901IHC] - Detection of human HIF-1 alpha by immunohistochemistry. Sample: FFPE section of renal cell carcinoma. Antibody: Affinity purified rabbit anti-HIF-1 alpha antibody (NB110-58773). Detection: DAB. HIF-1 alpha Antibody [NB100-449]

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-901IHC] - Immunofluorescence of wounded tissue and normal unwounded skin. Representative images for immunofluorescent staining of HIF-1 alpha on healed wounded skin and adjacent normal unwounded skin of wild type, heterozygous K14-Cre/homozygous floxed PHD-2, and heterozygous Col1 alpha2-Cre-ER/homozygous floxed PHD-2 mice at 400x magnification. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0093373), licensed under a CC-BY licence. HIF-1 alpha Antibody [NB100-479]

Immunohistochemistry: HIF-1 alpha Antibody Pack [NB100-901IHC] - In situ hybridization (antisense probe, Fig. 3a) and immunostaining with HIF1a Ab2 (Fig. 3c) on thin adjacent sections of NE-differentiated prostate adenocarcinoma showed co-localization of HIF1a1.2 transcript and HIF-1a protein. Incubation with sense probe did not generate any detectable hybridization signals (Fig. 3b). Both In situ hybridization (Fig. 3d antisense) and HIF-1a Ab2 immunostaining (Fig. 3f) were negative in non-NE-differentiated prostate adenocarcinoma. In situ hybridization with sense probe performed on non-NE-differentiated prostate cancer (Fig. 3e). Image collected and cropped by CiteAb from the following publication (//www.biomedcentral.com/1471-2407/10/385), licensed under a CC-BY licence. HIF-1 alpha Antibody (H1alpha67) [NB100-123]

Simple Western: HIF-1 alpha Antibody Pack [NB100-901IHC] - Simple Western lane view shows a specific band for HIF-1 alpha in 0.5 mg/ml of hypoxic HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system. HIF-1 alpha Antibody [NB100-449]

Knockdown Validated: HIF-1 alpha Antibody Pack [NB100-901IHC] - Morphological alterations induced by DMOG are HIF-1alpha dependent. glEND.2 cells were stably transfected with shGFP, shHIF-1 alpha or shHIF-2 alpha. The clones were cultured under normoxic (N) or hypoxic (H, 1% O2) conditions, or treated with DMOG for 6 h. 30 ug protein of cellular protein were loaded for the detection of HIF-1 alpha, and nuclear extracts (20 ug protein) were used for the detection of HIF-2 alpha. Image collected and cropped by CiteAb from the following publication (//biosignaling.biomedcentral.com/articles/10.1186/1478-811X-11-80) licensed under a CC-BY licence. HIF-1 alpha Antibody [NB100-449]

Knockdown Validated: HIF-1 alpha Antibody Pack [NB100-901IHC] - HIF-1 inhibits the apoptosis of hypoxic glioblastoma cells. U87MG cells were transfected with siRNA against HIF-1 alpha. Forty-eight hours after transfection, cells were incubated for 2 h in hypoxia (1% O2). HIF-1 alpha was detected by immunoblotting. beta-Actin was used as a loading control. The results are representative for three independent experiments. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/cddis2013562) licensed under a CC-BY licence. HIF-1 alpha Antibody (H1alpha67) [NB100-123]

• Reactivity: Hu, Mu, Rt
• Applications: WB, Simple Western, Flow, ICC/IF, IHC, IHC-P, IP, KD

HIF-1 alpha Antibody Pack Summary

• Immunogen: For NB100-131: Human HIF-1 alpha amino acids 329-530.
  For NB100-123: Fusion protein containing amino acids 432-528 of human HIF-1alpha.
  For NB100-449: peptide between residues 775 and the C-terminus (residue 826) of human HIF-1 alpha.
  For NB100-479: A fusion protein including amino acids 530 - 825 of the mouse HIF-1 alpha protein [Uniprot# Q61221].
• Specificity: These antibodies are specific for HIF-1 alpha.
• Gene: HIF1A

Applications/Dilutions

• Application Notes: Immunohistochemical staining of paraffin-embedded and frozen sections (only NB100-131 has been used on frozen sections). Antigen retrieval is strongly recommended. With the exception of NB100-449 and NB100-479 (which are also recommended for Western blot), these antibodies work for Western blot analysis but are not specifically recommended for this application. A starting dilution of 1:1000 is recommended with titration up or down depending upon the signal and background.
  See individual datasheets for validated applications.

Packaging, Storage & Formulations

• Storage: Store at 4C. Do not freeze.

Notes

This pack contains 1 vial each of: NB100-479SS (0.025 mL), NB100-449SS (0.025 mL), NB100-123SS (0.05 mL) and NB100-131SS (0.05 mL).

Alternate Names for HIF-1 alpha Antibody Pack

• 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. Antibody Packs are guaranteed for 1 year from date of receipt.

Publications for HIF-1 alpha Antibody Pack (NB100-901IHC)(5)

Publications using NB100-901IHC

• Lee, S, Kim, SJ, Kim, DJ et al. The Clinical Significanse of the Hypoxia Inducible Factor-1 C4d in Implantation Renal Biopsies Post-Transplant Biopsies. 22nd International Congress of the Transplantation Society. 10-Aug-08.
• Hill, P et al. Inhibition of hypoxia inducible factor hydroxylases protects against renal ischemia-reperfusion injury. J Am Soc Nephrol;19(1):39-46. 2008 Jan. [PMID: 18178798]
• Higami, Y et al. Energy restriction lowers the expression of genes linked to inflammation, the cytoskeleton, the extracellular matrix, and angiogenesis in mouse adipose tissue. J Nutr;136(2):343-52. 2006 Feb. [PMID: 16424110]
• Quintero, M et al. Nitric oxide is a factor in the stabilization of hypoxia-inducible factor-1alpha in cancer: role of free radical formation. Cancer Res;66(2):770-4. 2006 Jan 15. [PMID: 16424008]
• Tanaka, T et al. Hypoxia-inducible factor modulates tubular cell survival in cisplatin nephrotoxicity. Am J Physiol Renal Physiol289(5):F1123-33. 2005 Nov. [PMID: 15956779]

FAQs for HIF-1 alpha Antibody Pack (NB100-901IHC). (Showing 1 - 10 of 12 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 (https://www.novusbio.com/products/hif-1-alpha-antibody-h1alpha67_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:https://www.novusbio.com/support/hypoxia-and-hif-faqsAlso, 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: https://www.novusbio.com/support/hypoxia-and-hif-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 https://www.novusbio.com/support/hypoxia-and-hif-faqs(5)    Last but not the least, Novus technical support team may be contacted at: technical@novusbio.com 
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: http://www.novusbio.com/support/hypoxia-and-hif-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: https://www.novusbio.com/support/hypoxia-and-hif-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.
12. We have received an enquiry regarding HIF1 alpha antibody panel, product code NB 100-901IHC. I would appreciate if you are able to provide the WB testing protocol for this antibody.
    • There are four antibodies in this pack. We have specific western blot protocols for 3 of the 4 antibodies, as follows: Western Blot protocol specific for NB100-123, Western Blot protocol specific for NB100-479, Western Blot protocol specific for NB100-449. You may find our general western blot protocol useful.
Show All 12 FAQs.

Bioinformatics

• Gene Symbol: HIF1A