HIF-1 alpha Antibody (H1alpha67) - Azide and BSA Free

Images

 
Upregulation of HIF-1alpha in human PCa. Protein expression of HIF-1alpha, VEGF, and GLUT4 were examined with western blot, in PC-3, DU145, and LNCaP cells after various treatments as indicated. Data are expressed as ...read more
HIF-1 alpha was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line treated with DFOA using 1 ug/mL of mouse anti-HIF-1 alpha monoclonal antibody NB100-105. Cells were stained using a donkey ...read more
Analysis using the biotin conjugate of NB100-105. Staining of human glioblastoma multiforme.
Analysis using the Alexa Fluor (R) 488 conjugate of NB100-105. Staining of HIF-1 alpha in multiple myeloma cells: H929 cells (0.5x10^6) were stained with Alexa Fluor 488 (R) conjugated HIF-1 alpha antibody ...read more
Analysis of HIF-1 alpha in human hepatocytes from cancer patient using anti-HIF-1 alpha antibody NB100-105. Image from verified customer review. Image from the standard format of this antibody.
Analysis using the HRP conjugate of NB100-105. Detection of 50ug cobalt chloride induced COS-7 nuclear extracts (NB800-PC26) using NB100-105.
HIF-1 alpha induction by CoCl2 on Caki-1 cell lysate. Image from verified customer review. Image from the standard format of this antibody.
HIF1alpha/PFKFB3 is upregulated in beta-cells of HIP rats and humans with type 2 diabetes. Representative Western blot of PFKFB3 and HIF1alpha levels in nuclear-enriched- and whole cell extracts from non-diabetic (ND) ...read more
L. donovani infection induces HIF-1 alpha expression in CD11chi splenic DCs in an IRF-5 dependent manner. Mice were infected with 2x107 amastigotes intravenously. Immunoblot analysis of HIF-1 alpha expression in CD11c+ ...read more
Naive CD4 T cells from WT, VHL-deficient (Vhl KO), or HIF-1 alpha-deficient (HIF-1 alpha KO) mice were differentiated under IL-22-skewing conditions for a total of 60 h. Some cells remained at normoxia for the duration ...read more
Upregulation of HIF-1alpha in human Pca. HIF-1alpha protein was detected by western blot in nonmalignant (RWPE-1 and BPH1) and PCa cell lines (PC-3, DU145, LNCaP, and 22RV1) as indicated. Image collected and cropped by ...read more
HIF-1alpha expression in PDAC and adjacent normal pancreatic tissue. PDAC with weak (intensity 1) nuclear and cytoplasmic HIF-1alpha staining (left). PDAC with strong (intensity 3) nuclear and moderate (intensity 2) ...read more
Staining of HIF-1 alpha in human kidney using NB100-105. Renal tubular epithelium showed moderate membranous, cytoplasmic and nuclear staining, and glomeruli showed faint to moderate nuclear staining. Image from the ...read more
Analysis using the Alexa Fluor (R) 488 conjugate of NB100-105. Staining of HIF-1 alpha in H929 cells using HIF-1 alpha antibody. Image from verified customer review.
HIF-1 alpha was detected in immersion fixed DFO treated Hela cells (left) but was not detected in HIF-1 knockout HeLa cells (right) using Mouse Anti-human HIF-1 alpha monoclonal antibody (Catalog #NB100-105) at 25 ug/mL ...read more

Product Details

Summary
Reactivity Hu, Mu, Rt, Po, Bv, Ca, Fe, Ma, Pm, Pm, Rb, Sh, XpSpecies Glossary
Applications WB, ChIP, ELISA, Flow, Func, GS, ICC/IF, IHC, IP, NULL, WB, ChIP, CyTOF-ready, KD, KO
Clone
H1alpha67
Clonality
Monoclonal
Host
Mouse
Conjugate
Unconjugated
Format
Azide and BSA Free
Concentration
1 mg/ml

Order Details

HIF-1 alpha Antibody (H1alpha67) - Azide and BSA Free Summary

Immunogen
This HIF-1 alpha Antibody (H1alpha67) was developed against a fusion protein containing amino acids 432 - 528 of human HIF-1 alpha [Uniprot# Q16665].
Localization
HIF-1 is a nuclear protein
Isotype
IgG2b
Clonality
Monoclonal
Host
Mouse
Gene
HIF1A
Purity
Protein G purified
Innovator's Reward
Test in a species/application not listed above to receive a full credit towards a future purchase.

Applications/Dilutions

Dilutions
  • Chromatin Immunoprecipitation (ChIP) 1-5 ug/IP
  • Chromatin Immunoprecipitation 1 - 5 ug/IP. Use reported in scientific literature
  • CyTOF-ready
  • ELISA 1:100 - 1:2000. Use reported in scientific literature (PMID 20042684)
  • Flow Cytometry 1:10 - 1:1000
  • Gel Super Shift Assays 1:1 - 1:100. Use reported in scientific literature (PMID 22411794)
  • Immunoassay reported in scientific literature (PMID 26147748)
  • Immunoblotting reported in multiple pieces of scientific literature
  • Immunocytochemistry/ Immunofluorescence 1:50
  • Immunohistochemistry
  • Immunohistochemistry-Frozen 1:20 - 1:50
  • Immunohistochemistry-Paraffin 1:20 - 1:50
  • Immunoprecipitation 1:10 - 1:500
  • In vitro assay reported in multiple pieces of scientific literature
  • Knockdown Validated
  • Knockout Validated reported in scientific literature (PMID 26861754)
  • Ligand Activation reported in scientific literature (PMID 26147748)
  • Proximity Ligation Assay reported in scientific literature (PMID 27595394)
  • Tissue Culture Substratum
  • Western Blot
Application Notes
In WB, a band can be seen at 120 kDa representing HIF-1 alpha in induced tissues and cells. Multiple bands may be seen at 100-120 kDa representing post-translational modification of HIF-1 alpha. For WB, testing on nuclear extracts is recommended. We recommend the use of a highly sensitive ECL reagent, such as West Pico PLUS, for Western blot detection.
Theoretical MW
93 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Control
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
PC12 CoCl2 treated/Untreated Cell Lysate
PC12 Hypoxic / Normoxic Cell Lysate
Raw 264.7 Hypoxic / Normoxic Cell Lysate
L929 Hypoxic / Normoxic Cell Lysate
NIH 3T3 Hypoxic / Normoxic Cell Lysate

Reactivity Notes

Xenopus reactivity was reported in scientific literature (PMID: 18303027). Please note that this antibody is reactive to Mouse and derived from the same host, Mouse. Additional Mouse on Mouse blocking steps may be required for IHC and ICC experiments. Please contact Technical Support for more information. Canine reactivity reported in scientific literature (PMID: 24153191, 29974500). Feline reactivity reported in scientific literature (PMID: 30419801).

Packaging, Storage & Formulations

Storage
Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
Buffer
PBS
Preservative
No Preservative
Concentration
1 mg/ml
Purity
Protein G purified

Alternate Names for HIF-1 alpha Antibody (H1alpha67) - Azide and 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.

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Product General Protocols

Video Protocols

WB Video Protocol
ChIP Video Protocol
ChIP Webinar
ICC/IF Video Protocol

FAQs for HIF-1 alpha Antibody (NBP2-80760). (Showing 1 - 10 of 11 FAQ).

  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. Show All 11 FAQs.

Control Lysate(s)

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Blogs on HIF-1 alpha. Showing 1-10 of 37 blog posts - Show all blog posts.

Hypoxia-Dependent CAR Stabilizing Construct in T cells Improves Solid Tumor Targeting and Efficacy
By Victoria Osinski, PhDDespite advances in the development of cancer immunotherapies, those specifically targeting tumors still remains limited. Currently, there is great interest in utilizing chimeric antigen rece...  Read full blog post.

Tired T cells: Hypoxia Drives T cell Exhaustion in the Tumor Microenvironment
By Hunter MartinezThe paradigm shifting view of the immune system being leveraged to target cancer has led to numerous therapeutic breakthroughs. One major cell group responsible for this revelation is a T cell. ...  Read full blog post.

Understanding ‘Y’ in Breast Cancer: Crucial Role of DNA/RNA-binding Protein YB-1 in the Development, Pre-Invasive, and Metastatic Phases
Jamshed Arslan, Pharm D, PhD In the United States, 1 in 8 women will be diagnosed with breast cancer in her lifetime.1 Despite the prevalence, cancer genesis is a mystery. The heterogeneity of cancers makes it diff...  Read full blog post.


  Read full blog post.

Breast cancer stem cells survive chemotherapy through S100A10-ANXA2-SPT6 interaction that epigenetically promotes OCT4-mediated stemness
By Jamshed Arslan, Pharm D, PhDBreast cancer is the most common cancer among women that causes the greatest number of cancer-related deaths worldwide. After radiotherapy or cytotoxic chemotherapy like paclitax...  Read full blog post.

mTOR Signaling and the Tumor Microenvironment
By Yoskaly Lazo-Fernandez, PhD The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that, as a member of two distinct intracellular protein complexes, mTORC1 and mTORC2, regulates protein ...  Read full blog post.

Bad news for stomach cancer: BAMBI protein inhibits gastric carcinoma via TGF-beta/epithelial-mesenchymal transition signaling
By Jamshed Arslan Pharm.D. Gastric carcinoma is the second leading cause of cancer-related deaths worldwide. One of the key features of gastric carcinoma is acidosis, which promotes growth and metastasis of gastric ...  Read full blog post.

Developmental regulator Daam2 promotes glial cell tumors by degrading Von Hippel-Lindau protein
By Jamshed Arslan Pharm.D. Glioblastoma is an aggressive type of cancer that forms from the star-shaped glial cells of the central nervous system, called astrocytes. Intriguingly, several genes linked to glioblasto...  Read full blog post.

Stemness for Surviving Hypoxia: TGF-beta/Smad Signaling in Multiple Myeloma
By Jamshed Arslan Pharm.D. Multiple myeloma (MM) is a cancer of antibody-producing plasma cells. The bone marrow (BM) of MM patients is hypoxic, and MM cells overexpress many cancerous genes that are regulated by hy...  Read full blog post.

Forecasting and Targeting a Rare Cancer with Hypoxia-Inducible Factor
By Jamshed Arslan Pharm.D. Cancers of nerve, adipose, and other soft tissues are called soft tissue sarcomas (STS). Malignant peripheral nerve sheath tumor (MPNST) is an example of a rare and hard-to-treat STS; eve...  Read full blog post.

Showing 1-10 of 37 blog posts - Show all blog posts.
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Bioinformatics

Gene Symbol HIF1A