HIF-1 alpha Antibody

Images

 
Biological Strategies: Chromatin Immunoprecipitation: HIF-1 alpha Antibody [NB100-479] - ChIP-qPCR assay of HIF-1 binding to hypoxia response element (HRE) of the PKM2 gene in human MDA-MB-231 breast cancer cells ...read more
Western Blot: HIF-1 alpha Antibody [NB100-479] - Normoxic pre-treatment sensitizes glioma cells to radiation after graded chronic hypoxic (GCH) exposure. The results of anchorage-independent colony forming assays are ...read more
Immunohistochemistry: HIF-1 alpha Antibody [NB100-479] - Immunofluorescence of wounded tissue and normal unwounded skin. Representative images for immunofluorescent staining of HIF-1 alpha on healed wounded skin and ...read more
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-479] - Staining in normal mouse kidney showing (A) staining with antibody and (B) blocking peptide used at 10-fold excess concentration. Images provided by Dr ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-479] - Analysis of HIF-1 alpha in mouse hepatocytes (left lane: normoxic and right lane: hypoxic) using HIF-1 alpha antibody. Image from verified ...read more
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-479] - Analysis in paraffin section of mouse skin wound using NB100-479 at 1:6000. Image courtesy of product review by Ian Darby of RMIT University.
Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody [NB100-479] - Immunofluorescence (IF) labeling and confocal microscopy analysis of the distribution of HIF-1alpha (A1-D1) and Abeta (A2-D2) in APPsw cells. ...read more
Chromatin Immunoprecipitation: HIF-1 alpha Antibody [NB100-479] - MDA-MB-231 cells were exposed to 20% or 1% O2 for 16 hours, and chromatin immunoprecipitation (ChIP) was performed with the indicated antibody (Ab). ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-479] - Analysis in A. PC12 (rat) cells, 20% oxygen and B. PC12 (rat) cells, 1% oxygen.
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-479] - Analysis in a hypoxic sample. Lane 1: CoCl2 treated Cos-7 nuclear extract (50 ug, hypoxic). Lane 2: Untreated Cos-7 nuclear extract (50 ...read more
Western Blot: HIF-1 alpha Antibody [NB100-479] - U251 cells exposed to 4 hour hypoxia (0.5% oxygen) incubation. This image was submitted via a verified customer review.
Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody [NB100-479] - Detection of HIF-1 Alpha (green) in RCC4 cells using NB100-479. Nuclei (Blue) were counterstained using Hoechst 33258.
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-479] - Analysis of HIF-1 alpha in human kidney tumor.
Immunohistochemistry: HIF-1 alpha Antibody [NB100-479] - Staining of human placenta, villi.
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-479] - Effects of different routes of CDDP administration on remaining viable tumor tissue. Representative images of tumor tissue (i.v. CDDP, i.a. CDDP) stained ...read more
Immunohistochemistry: HIF-1 alpha Antibody [NB100-479] - Hif1alpha expression in preimplantation embryos. Co-staining of OCT4 and Hif1alpha in inner cell mass of blastocyst (top) and negative control images without ...read more

Product Details

Summary
Reactivity Hu, Mu, Rt, Po, Ca, Fi, Ha, Pm, RbSpecies Glossary
Applications WB, ChIP, Flow, IB, ICC/IF, IHC, IHC-Fr, IHC-P, IP, IHC-WhMt
Clonality
Polyclonal
Host
Rabbit
Conjugate
Unconjugated
Concentration
1.0 mg/ml
Validated by:
   

Biological Strategies

     

Order Details

HIF-1 alpha Antibody Summary

Immunogen
This HIF-1 alpha Antibody was developed against a fusion protein including amino acids 530 - 825 of the mouse HIF-1 alpha protein [Uniprot# Q61221].
Isotype
IgG
Clonality
Polyclonal
Host
Rabbit
Gene
HIF1A
Purity
Immunogen affinity purified
Innovator's Reward
Test in a species/application not listed above to receive a full credit towards a future purchase.

Applications/Dilutions

Dilutions
  • Western Blot
  • Chromatin Immunoprecipitation 1:10 - 1:500
  • Flow Cytometry 1:10 - 1:1000
  • Immunoblotting
  • Immunocytochemistry/Immunofluorescence 1:100
  • Immunohistochemistry 5-10 ug/ml
  • Immunohistochemistry-Frozen 5-10 ug/ml
  • Immunohistochemistry-Paraffin 5-10 ug/ml
  • Immunoprecipitation 1:10 - 1:500
  • Immunohistochemistry Whole-Mount
Application Notes
WB recognizes a band in hypoxic samples at ~115 kDa. HIF-1 alpha protein (NBC1-18422) has been successfully used for antibody preadsorption/blocking assay in IHC-P with HIF-1 alpha antibody NB100-479. Flow Cytometry usage reported in scientific literature (PMID 21917971). Use in Immunoblotting reported in scientific literature (PMID 28506759). Use in Immunohistochemistry-Whole mount reported in scientific literature (PMID: 27148974).
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
Human Pancreas Whole Tissue Lysate (Adult Whole Normal)
COS-7 Nuclear Hypoxic Induced Cell Lysate
Human Bone Marrow Whole Tissue Lysate (Adult Whole Diabetes)
HeLa CoCl2 treated/Untreated Cell Lysate
HepG2 CoCl2 treated/Untreated Cell Lysate
HeLa Hypoxic / Normoxic Cell Lysate
HepG2 Hypoxic / Normoxic Cell Lysate
HIF-1 alpha Knockout HeLa Cell Lysate
Control Peptide
HIF-1 alpha Recombinant Protein (NBC1-18422)
Reviewed Applications
Read 23 Reviews rated 4.4
using
NB100-479 in the following applications:

Publications
Read Publications using
NB100-479 in the following applications:

Reactivity Notes

Porcine reactivity reported in scientific literature (PMID: 31624943).

Packaging, Storage & Formulations

Storage
Store at 4C. Do not freeze.
Buffer
PBS
Preservative
0.05% Sodium Azide
Concentration
1.0 mg/ml
Purity
Immunogen affinity purified

Alternate Names for HIF-1 alpha Antibody

  • 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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Publications for HIF-1 alpha Antibody (NB100-479)(354)

We have publications tested in 9 confirmed species: Human, Mouse, Rat, Canine, Fish, Hamster, Moues, Porcine, Rabbit.

We have publications tested in 11 applications: ChIP, FLOW, IB, ICC/IF, IHC, IHC-Fr, IHC-P, IHC-WhMt, IP, WB, WB,ChIP.


Filter By Application
ChIP
(6)
FLOW
(2)
IB
(1)
ICC/IF
(29)
IHC
(30)
IHC-Fr
(6)
IHC-P
(34)
IHC-WhMt
(1)
IP
(6)
WB
(211)
WB,ChIP
(1)
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Human
(113)
Mouse
(140)
Rat
(20)
Canine
(1)
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(1)
Hamster
(2)
Moues
(1)
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(1)
Rabbit
(2)
All Species
Showing Publications 1 - 10 of 354. Show All 354 Publications.
Publications using NB100-479 Applications Species
Casciello F, Al-Ejeh F, Miranda M et al. G9a-mediated repression of CDH10 in hypoxia enhances breast tumour cell motility and associates with poor survival outcome Theranostics Mar 15 2020 [PMID: 32292512] (WB, Human) WB Human
Sharma M, Boytard L, Hadi T et Al. Enhanced glycolysis and HIF-1 alpha activation in adipose tissue macrophages sustains local and systemic interleukin-1 beta production in obesity Sci Rep Mar 27 2020 [PMID: 32221369] (IHC, Mouse) IHC Mouse
Fitzpatrick Sf, Lambden S, Macias D et Al. 2-Hydroxyglutarate Metabolism Is Altered in an in vivo Model of LPS Induced Endotoxemia Front Physiol Mar 3 2020 [PMID: 32194434] (WB, Human) WB Human
Ukeba D, Sudo H, Tsujimoto T et Al. Bone marrow mesenchymal stem cells combined with ultra-purified alginate gel as a regenerative therapeutic strategy after discectomy for degenerated intervertebral discs EBioMedicine Mar 3 2020 [PMID: 32143180] (IHC, Rabbit) IHC Rabbit
Burman A, Kropski JA, Calvi CL et al. Localized hypoxia links ER stress to lung fibrosis through induction of C/EBP homologous protein JCI Insight Aug 23 2018 [PMID: 30135303] (WB, Mouse) WB Mouse
Dai Z, Zhu MM, Peng Y et al. Therapeutic Targeting of Vascular Remodeling and Right Heart Failure in PAH with HIF-2alpha Inhibitor Am. J. Respir. Crit. Care Med. Jun 20 2018 [PMID: 29924941] (WB, Human) WB Human
Nascimento BPP, Bocco BMLC, Fernandes GW et al. Induction of type 2 iodothyronine deiodinase after status epilepticus modifies hippocampal gene expression in male mice Endocrinology Jun 13 2018 [PMID: 29905787] (Mouse) Mouse
Nakazeki F, Nishiga M, Horie T et al. Loss of periostin ameliorates adipose tissue inflammation and fibrosis in vivo Sci Rep Jun 4 2018 [PMID: 29867212] (WB, Mouse) WB Mouse
Chen A, Sceneay J, Godde N et al. Intermittent hypoxia induces a metastatic phenotype in breast cancer Oncogene May 1 2018 [PMID: 29713057] (Mouse) Mouse
Show All 354 Publications.

Reviews for HIF-1 alpha Antibody (NB100-479) (23) 4.423

Average Rating: 4.4
(Based on 23 reviews)
We have 23 reviews tested in 4 species: Human, Mouse, Rat, Human Breast Cancer Cell Lines and Human.

Reviews using NB100-479:
Filter by Applications
ChIP
(2)
WB
(17)
IHC-P
(4)
All Applications
Filter by Species
Human
(11)
Mouse
(9)
Rat
(2)
Human Breast Cancer Cell Lines and Human
(1)
All Species
Images Ratings Applications Species Date Details
Chromatin Immunoprecipitation HIF-1 alpha NB100-479
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reviewed by:
Anonymous
ChIP Human Breast Cancer Cell Lines and Human 05/17/2020
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Summary

ApplicationChromatin Immunoprecipitation
Sample TestedBreast cancer cells
SpeciesHuman Breast Cancer Cell Lines and Human
LotAN-1
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Anonymous
WB Human 04/16/2019
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Summary

ApplicationWestern Blot
Sample Tested16-HBE
SpeciesHuman
LotAL-6
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Anonymous
WB Human 09/18/2018
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Summary

ApplicationWestern Blot
Sample TestedHuman Cancer Cells
SpeciesHuman
LotAN-1
Immunohistochemistry-Paraffin HIF-1 alpha NB100-479
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reviewed by:
Anonymous
IHC-P Mouse 10/25/2017
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Summary

ApplicationImmunohistochemistry-Paraffin
Sample TestedIHC on parrafin sections (mouse)
SpeciesMouse
LotAK-4
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Kristina Cook
WB Human 02/23/2017
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Summary

ApplicationWestern Blot
Sample TestedU251 glioma cell line; whole cell lysate
SpeciesHuman

Comments

Comments4 hour hypoxia (0.5% oxygen) incubation
 
reviewed by:
Anonymous
WB Mouse 09/21/2016
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Summary

ApplicationWestern Blot
Sample TestedMouse kidney
SpeciesMouse

Blocking

Blocking DetailsBlocked with 5% Nonfat Dry Milk in 1x TBS-T, 1 hour at room temperature

Primary Anitbody

Dilution RatioDilution - 5% BSA in TBS-T solution buffer

Secondary Antibody

Secondary DescriptionHorse Anti-mouse IgG, HRP-linked Antibody
Secondary Manufacturer Cat##7076
Secondary Concentration2ul in 10ml of 5% Milk in TBS-T

Details

Detection NotesChemiluminescent detection
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Anonymous
WB Mouse 08/06/2016
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Summary

ApplicationWestern Blot
SpeciesMouse
FileView PDF
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Zhongxiao Wang
WB Mouse 10/05/2015
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Summary

ApplicationWestern Blot
Sample Testedmouse retina
SpeciesMouse
LotNB100-479

Blocking

Blocking Details5%milk/T-BST buffer, room temparature, 2hr

Primary Anitbody

Dilution Ratio1:1000 in 5%milk/T-BST buffer, 4 degree, overnight

Secondary Antibody

Secondary DescriptionGoat anti-rabbit IgG, HRP
Secondary Manufacturer Cat#Sigma AQ132P
Secondary Concentration1:3000

Details

Detection NotesDevelop machine, 30s exposure shows clear bands

Comments

CommentsThere's a non-specific band above for mouse retina samples
 
reviewed by:
Anonymous
WB Human 08/23/2015
View

Summary

ApplicationWestern Blot
Sample TestedHuman cancer cell whole cell lysate
SpeciesHuman

Blocking

Blocking DetailsBlocking with 5% non-fat milk for 30 min

Primary Anitbody

Dilution Ratio1:1000, overnight

Secondary Antibody

Secondary DescriptionDonkey anti-rabbit IgG, HRP
Secondary Manufacturer Cat#GE Healthcare, NA934V
Secondary Concentration1:2000

Details

Detection NotesRegular Protocol
 
reviewed by:
Anonymous
WB Human 03/27/2015
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Summary

ApplicationWestern Blot
Sample TestedHela lysates treated with MG132
SpeciesHuman

Blocking

Blocking Details5% milk in TBST

Primary Anitbody

Dilution Ratio1:2000

Secondary Antibody

Secondary DescriptionGoat anti-rabbit

Details

Detection Notesusing the regular pico from pierece, detection by Bio-rad imager, 1 min exposure time, DMSO as the NC, MG132 as the PC
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Anonymous
WB Mouse 02/18/2015
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Summary

ApplicationWestern Blot
Sample TestedMouse heart
SpeciesMouse
LotJ1

Blocking

Blocking Details5% milk in TBST for 1h at RT

Primary Anitbody

Dilution Ratio1:1000 in 5% milk TBST overnight at 4°C

Secondary Antibody

Secondary DescriptionGoat anti-rabbit IgG, HRP-coupled, 1:10 000 in 5% milk TBST, 1h at RT
Secondary Manufacturer Cat#AffiniPure Goat Anti-Rabbit IgG (H+L) from Jackson Immunolab
Secondary Concentration1:10 000

Details

Detection NotesECL Clarity, exposure time 30s

Comments

Comments- The signal was strong, it might be useful to decrease primary antibody concentration to reduce background noise.
 
reviewed by:
Anonymous
ChIP Human 12/15/2014
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Summary

ApplicationChromatin Immunoprecipitation
Sample TestedHEK293
SpeciesHuman
LotAA-1

Primary Anitbody

Dilution RatioUse 5ug antibody per 1ml lysate, and incubate at 4oC overnight

Details

Detection NotesReal-time PCR.
Fixation/Cross-LinkingElute the beads with 1% SDS, 0.1M NaHCO3 and then reverse the cross-linking by incubating at 65oC for 6 hours.
DNS SharingCovaris sonicator, 6 seconds for 200-500bp
Immunoprecipitation DetailsMillipore protein G magnetic beads 50ul/1ml lysate at room temperature for 10 minutes.
Crosslinking/Reversal Details1% formaldehyde for 5 minutes at room temperature
DNA Prurification DetailsQiagen PCR cleanup kit.
 
reviewed by:
Anonymous
WB Mouse 12/12/2014
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Summary

ApplicationWestern Blot
Sample TestedSee PMID 23232625
SpeciesMouse

Blocking

Blocking DetailsSee PMID 23232625

Details

Detection NotesSee PMID 23232625

Comments

CommentsThis antibody against HIF-1alpha reproducibly recognizes a single band in western blots assessing protein lysates from hypoxia-treated primary mouse aortic endothelial cells.Published in PMID: 23232625.
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Xiuquan Luo
WB Human 09/12/2014
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Summary

ApplicationWestern Blot
Sample TestedHEK293 cell lysate
SpeciesHuman
CommentsAntibody detects some non-specific bands, but give a good HIF1a signal at indicated MW.

Blocking

Blocking Details1xPBST with 5% non-fat milk, RT 1 hour

Primary Anitbody

Dilution Ratio1xPBST with 5% non-fat milk, overnight 4C, 1:1000 dilution

Secondary Antibody

Secondary DescriptionGoat anti-rabbit IgG-HRP
Secondary Manufacturer Cat#sc-2004
Secondary Concentration1:2000 dilution

Details

Detection NotesECL dura, exposured for 2-5 min

Comments

CommentsAntibody detects some non-specific bands, but give a good HIF1a signal at indicated MW.
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Ming Lu
WB Human 08/15/2014
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Summary

ApplicationWestern Blot
Sample TestedHuman Pancreatic Cancer Cell line
SpeciesHuman
LotAA
CommentsVery clean antibody, but it has multiple bands.

Blocking

Blocking Details2% cold fish geletin in TBS buffer, room temperature for 1hour

Primary Anitbody

Dilution Ratio1:1000 overnight at 4C in blocking buffer

Secondary Antibody

Secondary DescriptionGoat anti-rabbit IgG, HRP
Secondary Manufacturer Cat#Cell Signaling, #7074
Secondary Concentration1:5000

Details

Detection Notes1 min exposure shows band at correct MW.

Comments

CommentsVery clean antibody, but it has multiple bands.
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Molly Jenkins
WB Human 07/18/2014
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Summary

ApplicationWestern Blot
Sample TestedNuclear fraction, Human cell line
SpeciesHuman

Blocking

Blocking Details5% milk in TBST, 1hr at RT

Primary Anitbody

Dilution Ratio1:1000

Secondary Antibody

Secondary DescriptionGoat anti-rabbit HRP
Secondary Concentration1:20,000
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Jason Lee
WB Human 10/29/2013
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Summary

ApplicationWestern Blot
Sample TestedHuman MCF7 breast epithelial cells
SpeciesHuman
LotLot X-1
Commentsnuclear extract resolved in 8% SDS-PAGE gel

Blocking

Blocking Details3% BSA in TBST buffer 1 hour room temp

Primary Anitbody

Dilution Ratio1:2000 dilution in 3% BSA TBST 1 hour room temp

Secondary Antibody

Secondary Descriptiongoat anti-rabbit IgG HRP
Secondary Manufacturer Cat#Sigma
Secondary Concentration1:5000 in 5% skim milk TBST

Details

Detection NotesLAS4000 2 second exposure

Comments

Commentsnuclear extract resolved in 8% SDS-PAGE gel
Immunohistochemistry-Paraffin HIF-1 alpha NB100-479
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reviewed by:
Anonymous
IHC-P Mouse 06/27/2013
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Summary

ApplicationImmunohistochemistry-Paraffin
Sample Testedparaffin section of skin wound
SpeciesMouse
CommentsAntibody works much better when you find the necessary dilution which is quite low. Works best around 1:5000 to 1:7000.

Primary Anitbody

Dilution Ratio1:6000, room tempereature in Dako antibody diluent, overnight, 4oC

Secondary Antibody

Secondary Descriptionanti rabbit HRP polymer, Dako

Details

Fixation Details4% paraformaldehyde, DAKO heat mediated antigen retrieval, 20 mins, 98 deg C
Wash DescriptionDako wash buffer, Tris, contains Tween 20

Comments

CommentsAntibody works much better when you find the necessary dilution which is quite low. Works best around 1:5000 to 1:7000.
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Sandhya Khurana
WB Rat 02/05/2012
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Summary

ApplicationWestern Blot
Sample TestedPC12 nuclear extract, Sample Amount: 50ug
SpeciesRat

Blocking

Blocking DetailsBlocking Buffer: 5% Milk in TBST, Blocking Time: 30 minutes, Blocking Temp: Room temperature

Primary Anitbody

Dilution RatioDilution Ratio: 1:1000, Incubation Dilution Buffer: 5% Milk/TBST, Incubation Time: overnight, Incubation Temp: 4 C

Secondary Antibody

Secondary DescriptionSecondary Ab Dilution Ratio: 1:2000

Details

Detection NotesMethod:ECL, Time:2min, PosCtrl: Lysate from cells overexpressing HIF1a, NegCtrl: Untreated cells, Specific Bands:110-120kDa
 
reviewed by:
Anonymous
WB Rat 01/03/2012
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Summary

ApplicationWestern Blot
Sample TestedPC12 cell whole lysate, Sample Amount: 25ug
SpeciesRat
LotP1
CommentsThis antibody detected HIF-1a.

Blocking

Blocking DetailsBlocking Buffer: 5% Milk in TBST, Blocking Time: 1 hour, Blocking Temp: Room temperature

Primary Anitbody

Dilution RatioDilution Ratio: 1:1000, Incubation Dilution Buffer: blocking buffer, Incubation Time: overnight, Incubation Temp: 4 C

Comments

CommentsThis antibody detected HIF-1a.
Immunohistochemistry-Paraffin HIF-1 alpha NB100-479
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reviewed by:
Anonymous
IHC-P Mouse 07/30/2011
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Summary

ApplicationImmunohistochemistry-Paraffin
Sample Testednormal mouse kidney
SpeciesMouse
CommentsNuclear staining of (active) Hif-1alpha + cytoplasmic staining of (inactive) Hif-1alpha in normal mouse kidney (formalin-fixed, paraffin-embedded). No staining detected in controls. Staining done according to CSAII kit instructions. In addition, a permeabilization step was included (20 min. PBS + 0.5% Triton X-100) prior to Protein Block. Detection Method: DAB, Detection Time: 10 minutes, Positive Control: normal mouse kidney, Negative Control: rabbit isotype, antibody pre-absorbed with Hif-1alpha protein (NBC1-18422), Deparaffinization: 2x5 min. xylene, Antigen Retrieval: Target Retrieval Solution (Dako), 25 min. in pressure cooker

Blocking

Blocking DetailsBlocking Buffer: Protein Block (CSAII kit, Dako), Blocking Time: 15 minutes, Blocking Temp: room temperature

Primary Anitbody

Dilution RatioDilution Ratio: 1:20,000, Incubation Dilution Buffer:Ab Diluent, background reducing (Dako), Incubation Time/Temp:overnight @ 4C

Secondary Antibody

Secondary DescriptionSecondary Ab: CSAII Rabbit Link (Dako), Secondary Ab Dilution: 1:1
Secondary Manufacturer Cat#K1501

Details

Detection NotesPlease see Comments
Fixation DetailsPreparation: perfused PBS-formalin, postfixed overnight @ 4C in formalin, paraffin-embedded, Deparaffinization: 2x5 min. xylene

Comments

CommentsNuclear staining of (active) Hif-1alpha + cytoplasmic staining of (inactive) Hif-1alpha in normal mouse kidney (formalin-fixed, paraffin-embedded). No staining detected in controls. Staining done according to CSAII kit instructions. In addition, a permeabilization step was included (20 min. PBS + 0.5% Triton X-100) prior to Protein Block. Detection Method: DAB, Detection Time: 10 minutes, Positive Control: normal mouse kidney, Negative Control: rabbit isotype, antibody pre-absorbed with Hif-1alpha protein (NBC1-18422), Deparaffinization: 2x5 min. xylene, Antigen Retrieval: Target Retrieval Solution (Dako), 25 min. in pressure cooker
Immunohistochemistry-Paraffin HIF-1 alpha NB100-479
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reviewed by:
Paramahamsa Maturu
IHC-P Human 03/01/2011
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Summary

ApplicationImmunohistochemistry-Paraffin
Sample TestedHuman Kidney tumor
SpeciesHuman
LotI
CommentsThat works great without any background staining in the negative control. Detection Method: ABC and DAB, Positive Control: Glioblastoma, Negative Control: Without primary Ab, Deparaffinization: Xylen and ethanol, Antigen Retrieval: Tris -EDTA pH 9.0, 45 minutes at 95 degrees Celsius in water bath

Blocking

Blocking DetailsBlocking Buffer:.10% Normal Goat Serum in 1XPBS, Blocking Time: 1 hour, Blocking Temp: RT in humidified chamber

Primary Anitbody

Dilution RatioDilution Ratio: 1:100, Incubation Dilution Buffer: 2.5% Normal Goat Serum in 1X PBS, Incubation Time/Temp: overnight at 4C

Secondary Antibody

Secondary DescriptionSecondary Ab: Biotinylated Anti Rabbit IgG (H+L) made in Goat, Secondary Ab Dilution: 1:500
Secondary Manufacturer Cat#BA-1000

Details

Detection NotesPlease see Comments
Fixation DetailsSample Preparation: Paraffin embedded, Deparaffinization: Xylen and ethanol

Comments

CommentsThat works great without any background staining in the negative control. Detection Method: ABC and DAB, Positive Control: Glioblastoma, Negative Control: Without primary Ab, Deparaffinization: Xylen and ethanol, Antigen Retrieval: Tris -EDTA pH 9.0, 45 minutes at 95 degrees Celsius in water bath
Western Blot HIF-1 alpha NB100-479
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reviewed by:
JORCYK LAB
WB Mouse 02/01/2011
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Summary

ApplicationWestern Blot
Sample Tested4T1 cells whole cell lysate
SpeciesMouse
CommentsThe Hif1a band shows up even if the lysate concentration is very low. There are some background bands, but is acceptable, and there is quite a bit of lot to lot variations with this product... unfortunately.

Blocking

Blocking DetailsBlocking Buffer: 5% milk or 3% BSA, Blocking Time: overnight, Blocking Temp: 4 degrees Celsius

Primary Anitbody

Dilution RatioDilution Ratio: 1:1000-1:5000, Incubation Dilution Buffer: TBS-T or PBS-T, Incubation Time: 1 hr-overnight, Incubation Temp: 4C

Secondary Antibody

Secondary DescriptionSecondary Ab Dilution Ratio: 1:10,000-1:150,000

Details

Detection NotesMethod: ECL, Exposure Time: 10sec-10min, Pos.Ctrl: ECL reactive ladder, Bands:~120kDa(specific), ~80,55,72kDa(non-specific)

Comments

CommentsThe Hif1a band shows up even if the lysate concentration is very low. There are some background bands, but is acceptable, and there is quite a bit of lot to lot variations with this product... unfortunately.

Product General Protocols

Video Protocols

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

FAQs for HIF-1 alpha Antibody (NB100-479). (Showing 1 - 10 of 17 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. Show All 17 FAQs.

Control Lysate(s)

Secondary Antibodies

 

Isotype Controls

Other Available Formats

Alexa Fluor 350 NB100-479AF350
Alexa Fluor 405 NB100-479AF405
Alexa Fluor 488 NB100-479AF488
Alexa Fluor 594 NB100-479AF594
Alexa Fluor 647 NB100-479AF647
Alexa Fluor 700 NB100-479AF700
Alexa Fluor 750 NB100-479AF750
Allophycocyanin/Cy7 NB100-479APCCY7
Biotin NB100-479B
DyLight 350 NB100-479UV
DyLight 405 NB100-479V
DyLight 488 NB100-479G
DyLight 550 NB100-479R
DyLight 594 NB100-479DL594
DyLight 650 NB100-479C
DyLight 680 NB100-479FR
DyLight 755 NB100-479IR
FITC NB100-479F
HRP NB100-479H
Janelia Fluor 549 NB100-479JF549
Janelia Fluor 646 NB100-479JF646
PE/Atto594 NB100-479PEATT594
PE/Cy5.5 NB100-479PECY55
PE/Cy7 NB100-479PECY7

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Blogs on HIF-1 alpha. Showing 1-10 of 32 blog posts - Show all blog posts.

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.

The role of HIF-1 Alpha signaling in the retina under hypoxic conditions
Hypoxia inducible factor 1 (HIF-1) is a protein that plays an essential role in hypoxia, or low levels of cellular oxygen. HIF-1 is a heterodimeric protein that consists of a constitutively expressed beta subunit and oxygen related alpha subunit. ...  Read full blog post.

The relationship between Ki67 and HIF-1 in cancer
Ki67, also known as MKI67, is best known as the leading marker of cellular proliferation. Ki67 is regulated by a balance between synthesis and degradation, and often carries a very short half-life.  First discovered to be located to dividing cells,...  Read full blog post.

Controls for HIF-1 Alpha's WB, ICC-IF, IHC, IP & FLOW Analysis
Tips on positive and negative controls for HIF-1 alpha antibodies is one of the most Frequently Asked Questions on Hypoxia and HIFs. Here are top 5 suggestions from Novus Biologicals: The degradation of HIF1 alpha is the most common issue whic...  Read full blog post.

Understanding the relationship between HIF-1 alpha, Hypoxia and Epithelial-Mesenchymal Transition
Epithelial-mesenchymal transition (EMT) is a natural process by which epithelial cells lose their polarity and intercellular adhesion, and gain the migratory invasive properties of mesenchymal stem cells that can differentiate into a variety of cel...  Read full blog post.

HIF-2 alpha: HIF1A's Homologue with Similar and Divergent Functions
HIF-2 alpha is a member of the heterodimeric hypoxia-inducible factors/HIFs family (HIF-1, HIF-2, and HIF-3) which contains a common beta subunit but differ in their alpha subunits. Also called as EPAS1 or Mop2, HIF-2 alpha regulates cellular adapt...  Read full blog post.

Showing 1-10 of 32 blog posts - Show all blog posts.
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Bioinformatics

Gene Symbol HIF1A
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