Human/Mouse Total HIF-1 alpha/HIF1A DuoSet IC ELISA, 2 Plate

Lysates prepared from NIH-3T3 mouse embryonic fibroblast cells, untreated or treated with 150 µM of CoCl2, were incubated in wells coated with Human/Mouse Total HIF-1 alpha Capture Antibody. Unbound material was removed by washing, and bound material was solubilized in SDS gel sample buffer. Captured proteins were electrophoresed, transferred to PVDF membranes and immunoblotted with the Human/Mouse Total HIF-1 alpha Detection Antibody.

Lysates were prepared from MCF-7 human breast cancer cells, untreated or treated with 150 µM CoCl2. HIF-1 alpha was measured with the Human/Mouse Total HIF-1 alpha DuoSet IC ELISA and the same lysates were immunoblotted (inset) with Goat Anti-HIF-1 alpha Antibody (Catalog # AF1935). The DuoSet IC ELISA results correlate with the relative amounts of HIF-1 alpha detected by Western blot.

• Reactivity: Hu, Mu
• Applications: ELISA
• Conjugate: Biotin

Human/Mouse Total HIF-1 alpha/HIF1A DuoSet IC ELISA, 2 Plate Summary

• Source: N/A
• Assay Type: Solid Phase Sandwich ELISA
• Inter-Assay: See PDF Datasheet for details
• Intra-Assay: See PDF Datasheet for details
• Spike Recovery: See PDF Datasheet for details
• Sample Volume: See PDF Datasheet for details
• Gene: HIF1A

Applications/Dilutions

• Dilutions:
  • ELISA
• Application Notes: No significant interference observed with available related molecules.

Packaging, Storage & Formulations

• Storage: Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Human/Mouse Total HIF-1 alpha/HIF1A DuoSet IC ELISA, 2 Plate

• 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

The hypoxia-inducible transcription factor 1 alpha (HIF-1 alpha/HIF1A) is the regulated member of the transcription factor heterodimer HIF-1 alpha/beta. HIF-1 alpha/HIF1A binds to hypoxia-response elements (HREs) in the promoters of many genes involved in the adaptation to an environment of insufficient oxygen or hypoxia. Hypoxic tissue environments occur in vascular and pulmonary diseases as well as cancer, which illustrates the broad impact of gene regulation by HIF1A.

Publications for HIF-1 alpha (DYC1935-2)(34)

Publications using DYC1935-2

• Tojo, K;Yazawa, T; A high fraction of inspired oxygen does not mitigate atelectasis-induced lung tissue hypoxia or injury in experimental acute respiratory distress syndrome Scientific reports 2025-01-14 [PMID: 39809828] (Rat)
• Shams, S;Stilhano, RS;Silva, EA; Harnessing EGLN1 Gene Editing to Amplify HIF-1 alpha and Enhance Human Angiogenic Response bioRxiv : the preprint server for biology 2023-05-30 [PMID: 37398294] (Human)
• K P?oszczyca, M Chalimoniu, I Przybylska, M Czuba Effects of Short-Term Phosphate Loading on Aerobic Capacity under Acute Hypoxia in Cyclists: A Randomized, Placebo-Controlled, Crossover Study Nutrients, 2022-01-06;14(2):. 2022-01-06 [PMID: 35057416] (Human)
• M Jones-Muha, Q Shao, L Cain-Shiel, JP Shaffery, JP Warrington Acid Sensing Ion Channel 2a Is Reduced in the Reduced Uterine Perfusion Pressure Mouse Model and Increases Seizure Susceptibility in Pregnant Mice Cells, 2021-05-08;10(5):. 2021-05-08 [PMID: 34066660] (Mouse)
• J Filtjens, A Roger, L Quatrini, E Wieduwild, J Gouilly, G Hoeffel, R Rossignol, C Daher, G Debroas, S Henri, CM Jones, B Malissen, LK Mackay, A Moqrich, FR Carbone, S Ugolini Nociceptive sensory neurons promote CD8 T cell responses to HSV-1 infection Nature Communications, 2021-05-18;12(1):2936. 2021-05-18 [PMID: 34006861] (Mouse)
• M Okni?ska, B El-Hafny-R, A Paterek, U Mackiewicz, C Crola-Da S, K Brodaczews, M M?czewski, C Kieda Treatment of hypoxia-dependent cardiovascular diseases by myo-inositol trispyrophosphate (ITPP)-enhancement of oxygen delivery by red blood cells J. Cell. Mol. Med., 2020-01-19;0(0):. 2020-01-19 [PMID: 31957267] (Mouse)
• Wei Shan, Yunfei Wang, Zhen Zhang, Jie Xing, Jienan Xu, Wen Xiao et al. Qingre Baidu mixture‑induced effect of AI‑2 on Staphylococcus�aureus and Pseudomonas�aeruginosa biofilms in chronic and refractory wounds Experimental and Therapeutic Medicine 2019-03-13 [PMID: 30988710] (Bacteria)
• AN Billin, SE Honeycutt, AV McDougal, JP Kerr, Z Chen, JM Freudenber, DK Rajpal, G Luo, HF Kramer, RS Geske, F Fang, B Yao, RV Clark, J Lepore, A Cobitz, R Miller, K Nosaka, AC Hinken, AJ Russell HIF prolyl hydroxylase inhibition protects skeletal muscle from eccentric contraction-induced injury Skelet Muscle, 2018-11-13;8(1):35. 2018-11-13 [PMID: 30424786] (Mouse)
• M Li, Y Cui, W He, X Deng, Y Wang, M Cai, Y Wang, J Pei, X Mei, P Wu Effects of Triple-Mutated Hypoxia-Inducible Factor-1? on Angiogenesis and Cardiac Function Improvement in Rats with Myocardial Infarction Cell. Physiol. Biochem., 2018-11-13;50(6):2329-2340. 2018-11-13 [PMID: 30423574] (Rat)
• A Sturrock, D Woller, A Freeman, K Sanders, R Paine Consequences of Hypoxia for the Pulmonary Alveolar Epithelial Cell Innate Immune Response J. Immunol., 2018-10-31;0(0):. 2018-10-31 [PMID: 30381478] (Mouse)
• R Terao, M Honjo, M Aihara Apolipoprotein M Inhibits Angiogenic and Inflammatory Response by Sphingosine 1-Phosphate on Retinal Pigment Epithelium Cells Int J Mol Sci, 2017-12-31;19(1):. 2017-12-31 [PMID: 29301231] (Human)
• Y Guo Role of HIF-1a in regulating autophagic cell survival during cerebral ischemia reperfusion in rats Oncotarget, 2017-10-01;8(58):98482-98494. 2017-10-01 [PMID: 29228704] (Human, Rat)
• J Miku?a-Pie, P Uruski, S Szubert, K Maksin, R Moszy?ski, D Szpurek, A Wo?niak, S Sajdak, A Tykarski, K Ksi??ek The Proangiogenic Capabilities of Malignant Ascites Generated by Aggressive Ovarian Tumors Biomed Res Int, 2017-09-20;2017(0):2592496. 2017-09-20 [PMID: 29085834] (Human)
• K Wang, B Peng, J Xiao, O Weinreb, MBH Youdim, B Lin Iron-Chelating Drugs Enhance Cone Photoreceptor Survival in a Mouse Model of Retinitis Pigmentosa Invest. Ophthalmol. Vis. Sci., 2017-10-01;58(12):5287-5297. 2017-10-01 [PMID: 29049732] (Mouse)
• YJ Kim, SJ Park, NR Kim, HS Chin Effects of Histone Deacetylase Inhibitor (Valproic Acid) on the Expression of Hypoxia-inducible Factor-1 Alpha in Human Retinal M�ller Cells Korean J Ophthalmol, 2017-02-02;31(1):80-85. 2017-02-02 [PMID: 28243027] (Human)
• VEGF released by deferoxamine preconditioned mesenchymal stem cells seeded on collagen-GAG substrates enhances neovascularization Sci Rep, 2016-11-10;6(0):36879. 2016-11-10 [PMID: 27830734] (Human)
• Yang Xu Glycolysis determines dichotomous regulation of T cell subsets in hypoxia J Clin Invest, 2016-06-13;0(0):. 2016-06-13 [PMID: 27294526] (Human)
• Nunes D, Dias-Neto E, Cardo-Vila M, Edwards J, Dobroff A, Giordano R, Mandelin J, Brentani H, Hasselgren C, Yao V, Marchio S, Pereira C, Passetti F, Calin G, Sidman R, Arap W, Pasqualini R Synchronous down-modulation of miR-17 family members is an early causative event in the retinal angiogenic switch. Proc Natl Acad Sci U S A, 2015-03-09;112(12):3770-5. 2015-03-09 [PMID: 25775553] (Human)
• Chen , Yulong, Terajima , Masahiko, Yang , Yanan, Sun , Li, Ahn , Young-Ho, Pankova , Daniela, Puperi , Daniel S, Watanabe , Takeshi, Kim , Min P, Blackmon , Shanda H, Rodriguez , Jaime, Liu , Hui, Behrens , Carmen, Wistuba , Ignacio, Minelli , Rosalba, Scott , Kenneth, Sanchez-Adams , Johannah, Guilak , Farshid, Pati , Debanand, Thilaganathan , Nishan, Burns , Alan R, Creighton , Chad J, Martinez , Elisabet, Zal , Tomasz, Grande-Allen , K Jane, Yamauchi , Mitsuo, Kurie , Jonathan Lysyl hydroxylase 2 induces a collagen cross-link switch in tumor stroma. J Clin Invest, 2015-02-09;125(3):1147-62. 2015-02-09 [PMID: 25664850] (Human)
• Braicu E, Luketina H, Richter R, Cacsire Castillo-Tong D, Lambrechts S, Mahner S, Concin N, Mentze M, Zeillinger R, Vergote I, Sehouli J HIF1alpha is an independent prognostic factor for overall survival in advanced primary epithelial ovarian cancer - a study of the OVCAD Consortium. Onco Targets Ther, 2014-09-11;7(0):1563-9. 2014-09-11 [PMID: 25246800] (Human)
• Chawla S, Rahar B, Singh M, Bansal A, Saraswat D, Saxena S Exogenous sphingosine-1-phosphate boosts acclimatization in rats exposed to acute hypobaric hypoxia: assessment of haematological and metabolic effects. PLoS ONE, 2014-06-02;9(6):e98025. 2014-06-02 [PMID: 24887065] (Rat)
• Park S, Kim B, Lee J, Park S, Lee S, Dong S, Rho S GABARBP down-regulates HIF-1alpha expression through the VEGFR-2 and PI3K/mTOR/4E-BP1 pathways. Cell Signal, 2014-03-29;26(7):1506-13. 2014-03-29 [PMID: 24686084] (Human)
• Koch K, Refaian N, Hos D, Schlereth S, Bosch J, Cursiefen C, Heindl L Autocrine impact of VEGF-A on uveal melanoma cells. Invest Ophthalmol Vis Sci, 2014-04-25;55(4):2697-704. 2014-04-25 [PMID: 24677103] (Human)
• Khong T, Thairu N, Larsen H, Dawson P, Kiriakidis S, Paleolog E Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer. BMC Cancer, 2013-11-02;13(0):518. 2013-11-02 [PMID: 24180698] (Human)
• Lee S, No Y, Dang D, Dang L, Yang V, Shim H, Yun C Regulation of hypoxia-inducible factor 1alpha (HIF-1alpha) by lysophosphatidic acid is dependent on interplay between p53 and Kruppel-like factor 5. J Biol Chem, 2013-07-23;288(35):25244-53. 2013-07-23 [PMID: 23880760] (Human)
• Hamed E, El-Abaseri T, Mohamed A, Ahmed A, El-Metwally T Hypoxia and oxidative stress markers in pediatric patients undergoing hemodialysis: cross section study. BMC Nephrol, 2012-10-13;13(0):136. 2012-10-13 [PMID: 23061474] (Human)
• Lange CA, Luhmann UF, Mowat FM Von Hippel-Lindau protein in the RPE is essential for normal ocular growth and vascular development. Development, 2012-05-23;139(13):2340-50. 2012-05-23 [PMID: 22627278] (Mouse)
• Dungwa JV, Hunt LP, Ramani P Overexpression of carbonic anhydrase and HIF-1alpha in Wilms tumours. BMC Cancer, 2011-09-12;11(0):390. 2011-09-12 [PMID: 21910893] (Human)
• Hindryckx P, De Vos M, Jacques P, Ferdinande L, Peeters H, Olievier K, Bogaert S, Brinkman B, Vandenabeele P, Elewaut D, Laukens D Hydroxylase inhibition abrogates TNF-alpha-induced intestinal epithelial damage by hypoxia-inducible factor-1-dependent repression of FADD. J. Immunol., 2010-10-13;185(10):6306-16. 2010-10-13 [PMID: 20943999] (Mouse)
• Xu L, Nilsson MB, Saintigny P, Cascone T, Herynk MH, Du Z, Nikolinakos PG, Yang Y, Prudkin L, Liu D, Lee JJ, Johnson FM, Wong KK, Girard L, Gazdar AF, Minna JD, Kurie JM, Wistuba II, Heymach JV Epidermal growth factor receptor regulates MET levels and invasiveness through hypoxia-inducible factor-1alpha in non-small cell lung cancer cells. Oncogene, 2010-02-15;29(18):2616-27. 2010-02-15 [PMID: 20154724] (Human)
• Guo S, Miyake M, Liu KJ, Shi H Specific inhibition of hypoxia inducible factor 1 exaggerates cell injury induced by in vitro ischemia through deteriorating cellular redox environment. J. Neurochem., 2009-01-29;108(5):1309-21. 2009-01-29 [PMID: 19183269] (Human)
• Chung I, Han G, Seshadri M, Gillard BM, Yu WD, Foster BA, Trump DL, Johnson CS Role of vitamin D receptor in the antiproliferative effects of calcitriol in tumor-derived endothelial cells and tumor angiogenesis in vivo. Cancer Res., 2009-01-13;69(3):967-75. 2009-01-13 [PMID: 19141646] (Mouse)
• Wang B, Wood IS, Trayhurn P Hypoxia induces leptin gene expression and secretion in human preadipocytes: differential effects of hypoxia on adipokine expression by preadipocytes. J. Endocrinol., 2008-05-07;198(1):127-34. 2008-05-07 [PMID: 18463145] (Human)
• Fritz WA, Lin TM, Peterson RE The aryl hydrocarbon receptor (AhR) inhibits vanadate-induced vascular endothelial growth factor (VEGF) production in TRAMP prostates. Carcinogenesis, 2008-03-20;29(5):1077-82. 2008-03-20 [PMID: 18359762] (Mouse)

FAQs for HIF-1 alpha (DYC1935-2). (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