HIF-1 alpha Antibody

Images

 
Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody [NB100-134] - Hif1alpha expression in 9.5dpc primordial germ cells. Whole-mount staining of Hif1alpha and GFP in Oct4-GFP embryo (lateral view). Scale bar: ...read more
Biological Strategies: Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody [NB100-134] - Analysis of HIF-1 alpha in Normoxic (N) and Hypoxic (H) ARPE-19 cells using anti-HIF-1 alpha antibody NB100-134. ...read more
Western Blot: HIF-1 alpha Antibody [NB100-134] - Analysis of HIF-1 alpha in overexpression and endogenous HIF-1 alpha & HIF-2 alpha using anti-HIF-1 alpha antibody NB100-134. The data showed that HIF-1 alpha antibody ...read more
Orthogonal Strategies: Dual RNAscope ISH-IHC: HIF-1 alpha Antibody [NB100-134] - Formalin-fixed paraffin-embedded tissue sections of human kidney were probed for HIF-1 alpha mRNA (ACD RNAScope Probe, catalog ...read more
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-134] - Staining in the canine CL on days 10 to 70 after ovulation. PC = positive control (human placenta). NC = negative control. Image from verified customer ...read more
Simple Western: HIF-1 alpha Antibody [NB100-134] - Image shows a specific band for HIF-1 alpha in 0.2 mg/mL of Hypoxic HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa ...read more
Biological Strategies: Immunoprecipitation: HIF-1 alpha Antibody [NB100-134] - PC-3 cell lysates run with cobalt chloride treatments. Immunoprecipitation was performed with protein A/G agarose beads. This data ...read more
Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody [NB100-134] - Hif1alpha expression in neonatal and adult ovary. Section of P5 ovary showing MVH expression in all oocytes and Hif1alpha expression only in ...read more
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-134] - Analysis of HIF-1 alpha in zebrafish retina tissue using NB100-134 HIF-1 alpha antibody. Mouse tissue was used as a control. Image from verified customer ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-134] - Analysis of HIF-1 alpha in human retinal and choroidal primary endothelia cells exposed to hypoxic conditions in the times noted using ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-134] - Analysis of HIF-1 alpha using NB100-134, on normoxic and hypoxic nuclear rat cell lysates.
Immunohistochemistry: HIF-1 alpha Antibody [NB100-134] - Staining of human kidney, renal tubular epithelium in cortex using NB100-134.
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-134] - Analysis of FFPE tissue section of human endometrium carcinoma AN3CA cell line based xenograft using rabbit polyclonal HIF-1 alpha antibody NB100-134 at ...read more

Product Details

Summary
Reactivity Hu, Mu, Rt, Ca, Gp, Pm, Xp, ZeSpecies Glossary
Applications WB, Simple Western, ELISA, GS, IB, ICC/IF, IHC, IHC-Fr, IHC-P, IP, ChIP, Dual ISH-IHC, KO
Clonality
Polyclonal
Host
Rabbit
Conjugate
Unconjugated
Concentration
1.0 mg/ml

HIF-1 alpha Antibody Summary

Immunogen
This HIF-1 alpha Antibody was developed against a fusion protein made to an internal sequence of human HIF-1 alpha (containing amino acids 432 - 528) [Uniprot# Q16665].
Localization
Nucleus
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 1:500 - 1:1000
  • Simple Western 1:100
  • ELISA
  • Gel Super Shift Assays 1:1 - 1:100
  • Immunoblotting
  • Immunocytochemistry/Immunofluorescence 1:100 - 1:500
  • Immunohistochemistry 1:100 - 1:500
  • Immunohistochemistry-Frozen 1:100 - 1:500
  • Immunohistochemistry-Paraffin 1:100 - 1:500
  • Immunoprecipitation 1:1000
  • Chromatin Immunoprecipitation (ChIP)
  • Dual RNAscope ISH-IHC
  • Knockout Validated
Application Notes
Use in chromatin immunoprecipitation reported in scientific literature (PMID: 30537732). ICC/IF usage was reported in scientific literature (PMID: 21270095, 21270095). In WB a band is observed approx. 120 kDa. IHC applications have been mixed on Mouse. For ChIP, nuclear extracts are recommended. Prior to immunostaining paraffin tissues, antigen retrieval with sodium citrate buffer (pH 6.0) is recommended. In Simple Western only 10 - 15 uL of the recommended dilution is used per data point. Knock Out Validation was reported in scientific literature (PMID: 26861754). Use in Immunoblotting reported in multiple pieces of scientific literature.Use in ELISA reported in scientific literature (PMID: 24837709).
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
COS-7 Nuclear Hypoxic Induced Cell Lysate
Human Placenta Whole Tissue Lysate (Adult Whole Normal)
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
Reviewed Applications
Read 14 Reviews rated 4.8
using
NB100-134 in the following applications:

Publications
Read Publications using
NB100-134 in the following applications:

Reactivity Notes

Canine reactivity was reported in scientific literature (PMID: 24140705). Mouse reactivity results are mixed. Cross-reactivity with Zebrafish was reported via a customer review.

Packaging, Storage & Formulations

Storage
Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
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.

Customers Who Viewed This Item Also Viewed...

NBP2-88941
Species: Hu
Applications: IHC, IHC-P
NB100-124
Species: Hu, Mu, Rt, Bv, Ma, Pm, Sh
Applications: WB, ChIP, GS, IB, ICC/IF, IHC, IHC-P, IP, CHIP-SEQ
H00007428-D01P
Species: Hu, Mu
Applications: WB
NB100-122
Species: Hu, Mu, Rt, Fi, Ha, Pm, Rb, Re, Sh
Applications: WB, Simple Western, ChIP, ELISA, Flow, GS, IB, ICC/IF, IHC, IHC-Fr, IHC-P, IP, In vitro, PAGE, Dual ISH-IHC, KD, KO
AF887
Species: Hu, Mu, Rt
Applications: WB, Simple Western, IHC, CyTOF-ready, ICFlow
NB110-39113
Species: Hu, Mu, Rt, Rb
Applications: WB, ChIP, Flow, ICC/IF, IHC, IHC-Fr, IHC-P, In vitro, Flow-IC
NB100-417
Species: Hu, Mu, Rt, Pl
Applications: WB, Simple Western, ChIP, ELISA, Flow, GS, IB, ICC/IF, IHC, IHC-Fr, IHC-P, MiAr, Dual ISH-IHC, KO
NB200-103
Species: Hu, Mu, Rt, Ch, Ye, Xp(-)
Applications: WB, ELISA, Flow, ICC/IF, IHC, IHC-Fr, IHC-P, IP, CyTOF-ready, Flow-IC
NBP2-22203
Species: Hu, Mu, Rt, Pm
Applications: WB, ELISA, Flow, ICC/IF, IHC, IHC-P
NBP2-67360
Species: Hu, Mu, Rt
Applications: WB, Flow, ICC/IF, IHC, IHC-P, IP
NB600-607
Species: Hu, Rt
Applications: WB, ELISA, IHC, IHC-P
NB100-105
Species: Hu, Mu, Rt, Po, Bv, Ca, Fe, Ma, Pm, Pm, Rb, Sh, Xp
Applications: WB, ChIP, ELISA, Flow, GS, IA, IB, ICC/IF, IHC, IHC-Fr, IHC-P, IP, In vitro, PLA, TCS, KO, LA
NB300-605
Species: Hu, Mu, Rt, Po
Applications: WB, Flow, IB, ICC/IF, IHC, IHC-P, In vitro
NB100-137
Species: Hu, Mu, Rt, Pm
Applications: WB, Simple Western, Flow, ICC/IF, IHC, IHC-P, IP, KD

Publications for HIF-1 alpha Antibody (NB100-134)(170)

We have publications tested in 6 confirmed species: Human, Mouse, Rat, Canine, Xenopus, Zebrafish.

We have publications tested in 9 applications: ChIP, ChIp, ICC/IF, IHC, IHC-Fr, IHC-P, IP, Simple Western, WB.


Filter By Application
ChIP
(40)
ChIp
(1)
ICC/IF
(14)
IHC
(20)
IHC-Fr
(4)
IHC-P
(10)
IP
(6)
Simple Western
(1)
WB
(81)
All Applications
Filter By Species
Human
(78)
Mouse
(45)
Rat
(16)
Canine
(2)
Xenopus
(1)
Zebrafish
(1)
All Species
Showing Publications 1 - 10 of 170. Show All 170 Publications.
Publications using NB100-134 Applications Species
Dowdell AS, Cartwright IM, Goldberg MS et al. The HIF target ATG9A is Essential for Epithelial Barrier Function and Tight Junction Biogenesis Mol. Biol. Cell Jul 29 2020 [PMID: 32726170] (IP) IP
Cohrs G, Drucks B, Synowitz M et al. Expression patterns of hypoxia inducible factors, pro-inflammatory, and neuroprotective cytokines in neuroepithelial tissues of lumbar spinal lipomas - a pilot study World Neurosurg Jun 6 2020 [PMID: 32522652] (IHC, Human) IHC Human
James NE, Emerson JB, Borgstadt AD et al. The biomarker HE4 (WFDC2) promotes a pro-angiogenic and immunosuppressive tumor microenvironment via regulation of STAT3 target genes Sci Rep May 22 2020 [PMID: 32444701] (WB, Human) WB Human
MEnEgaut L, Thomas C, Jalil A et al. Interplay between Liver X Receptor and Hypoxia Inducible Factor 1 alpha Potentiates Interleukin-1 beta Production in Human Macrophages Cell Rep Jan 1 2020 [PMID: 32433974] (ICC/IF, Mouse) ICC/IF Mouse
Peng W, de Bruijn HS, Ten Hagen TLM et al. Targeted Photodynamic Therapy of Human Head and Neck Squamous Cell Carcinoma with Anti-epidermal Growth Factor Receptor Antibody Cetuximab and Photosensitizer IR700DX in the Mouse Skin-fold Window Chamber Model Photochem. Photobiol. Mar 29 2020 [PMID: 32222965] (IHC, Mouse) IHC Mouse
Liu L, Zhang L, Zhao J et Al. Tumor Necrosis Factor Receptor-Associated Protein 1 Protects against Mitochondrial Injury by Preventing High Glucose-Induced mPTP Opening in Diabetes Oxid Med Cell Longev Mar 6 2020 [PMID: 32215175]
Lucia K, Wu Y, Garcia JM et al. Hypoxia and the hypoxia inducible factor 1 alpha activate protein kinase A by repressing RII beta subunit transcription Oncogene Apr 1 2020 [PMID: 32111982] (WB, IHC, IP, ChIP, Rat) WB, IHC, IP, ChIP Rat
Andrade FO, Nguyen NM, Warri A, Hilakivi-Clarke L Reversal of increased mammary tumorigenesis by valproic acid and hydralazine in offspring of dams fed high fat diet during pregnancy Sci Rep Dec 30 2019 [PMID: 31889127] (WB, Mouse) WB Mouse
Chaboissier MC, Richardson N, Gillot I, Gregoire EP Roles of Id1/HIF-1 and CDK5/HIF-1 in cell cycle reentry induced by amyloid-beta peptide in post-mitotic cortical neuron Biochim Biophys Acta Mol Cell Res Dec 26 2019 [PMID: 31884068] (WB, Rat) WB Rat
Zhao L, Zhao J, Gao R, et al. Intrabody against prolyl hydroxylase 2 ameliorates acetaminophen-induced acute liver injury in mice via concomitant promotion of angiogenesis and redox homeostasis Biomed. Pharmacother. Dec 16 2019 [PMID: 31855737] (IHC-P, Mouse) IHC-P Mouse
Show All 170 Publications.

Reviews for HIF-1 alpha Antibody (NB100-134) (14) 4.814

Average Rating: 4.8
(Based on 14 reviews)
We have 14 reviews tested in 4 species: Human, Rat, Other, Zebrafish
.

Reviews using NB100-134:
Filter by Applications
WB
(10)
IHC-P
(2)
IF
(1)
IP
(1)
All Applications
Filter by Species
Human
(9)
Rat
(2)
Other
(2)
Zebrafish
(1)
All Species
Images Ratings Applications Species Date Details
Western Blot HIF-1 alpha NB100-134
Enlarge
reviewed by:
KARTHIKEYAN THIRUGNANAM
WB Zebrafish
 02/07/2020
View

Summary

ApplicationWestern Blot
Sample TestedZebrafish embryo lysates
SpeciesZebrafish
LotAP-13
Western Blot HIF-1 alpha NB100-134
Enlarge
reviewed by:
Anonymous
WB Human 10/28/2018
View

Summary

ApplicationWestern Blot
Sample TestedHuman MD-MB-231 cells,bt549
SpeciesHuman
LotAE-7
Western Blot HIF-1 alpha NB100-134
Enlarge
reviewed by:
Anonymous
WB Human 04/30/2018
View

Summary

ApplicationWestern Blot
Sample Tested20 ug whole cell lysate
SpeciesHuman
LotAE-3
Western Blot HIF-1 alpha NB100-134
Enlarge
reviewed by:
Anonymous
WB Human 03/22/2017
View

Summary

ApplicationWestern Blot
Sample TestedHCT116 whole cell lysate
SpeciesHuman
LotAA2
Immunohistochemistry-Paraffin HIF-1 alpha NB100-134
Enlarge
reviewed by:
Helder Andre
IHC-P Other 08/02/2016
View

Summary

ApplicationImmunohistochemistry-Paraffin
Sample TestedZebrafish retina with mouse tissue as control
SpeciesOther
Western Blot HIF-1 alpha NB100-134
Enlarge
reviewed by:
Helder Andre
WB Human 07/07/2016
View

Summary

ApplicationWestern Blot
Sample TestedRetinal and Choroidal primary endothelia
SpeciesHuman

Blocking

Blocking Details5% milk/TBS, 1h, RT

Primary Anitbody

Dilution Ratio1:250 in 1% milk/TBS, ON, 4C

Secondary Antibody

Secondary Descriptionswine anti-rabbit IgG-HRP
Secondary Concentration1:5000 in 1% milk/TBS, 1h, RT

Details

Detection NotesECL+
 
reviewed by:
Darren Richard
WB Rat 07/23/2015
View

Summary

ApplicationWestern Blot
Sample TestedVascular Smooth Muscle Cells
SpeciesRat

Blocking

Blocking DetailsTBS, Milk, 2hrs

Primary Anitbody

Dilution Ratio1:1000

Secondary Antibody

Secondary DescriptionGoat anti mouse, Promega
Secondary Concentration1:3000

Details

Detection NotesECL, homemade

Comments

CommentsA good choice for Westerns
Western Blot HIF-1 alpha NB100-134
Enlarge
reviewed by:
Anonymous
WB Human 06/01/2015
View

Summary

ApplicationWestern Blot
Sample TestedMDA-MB-231 Cell Lysate
SpeciesHuman

Blocking

Blocking Details5% milk

Primary Anitbody

Dilution Ratio1:1000

Secondary Antibody

Secondary Descriptiondonkey anti-rabbit IgG, HRP
Secondary Manufacturer Cat#Amersham NA934V
Secondary Concentration1:3000

Details

Detection NotesBIO RAD clarity western ECL (Antibody specificity; not react to HIF2a-overexpression as shown in the picture)
Immunofluorescence HIF-1 alpha NB100-134
Enlarge
reviewed by:
Helder Andre
IF Human 05/14/2015
View

Summary

ApplicationImmunofluorescence
Sample TestedARPE-19 cells
SpeciesHuman

Blocking

Blocking Details10% goat serum in PBS-TritonX (0.1%) (30min @ RT)

Primary Anitbody

Dilution Ratio1:500 in blocking solution (1h @ RT)

Secondary Antibody

Secondary Description1:1000 Goat anti-rabbit IgG-A488 in blocking solution (1h @ RT)

Details

Detection NotesEpifluorescence microscope
Fixation DetailsFixed in 4% buffered formalin (15min @ RT); Permeabilized in PBS-TritonX (0.5%) (10min @ RT)
Wash Description3x5min PBS
Western Blot HIF-1 alpha NB100-134
Enlarge
reviewed by:
Xiuquan Luo
WB Human 09/18/2014
View

Summary

ApplicationWestern Blot
Sample TestedHEK293 cell lysate
SpeciesHuman
CommentsThere are some non-specific bands, but the HIF1alpha bands are strong and clear.

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

CommentsThere are some non-specific bands, but the HIF1alpha bands are strong and clear.
Western Blot HIF-1 alpha NB100-134
Enlarge
reviewed by:
Pramod Mallikarjuna
WB Human 08/30/2014
View

Summary

ApplicationWestern Blot
Sample TestedClear cell renal cell carcinoma samples
SpeciesHuman

Blocking

Blocking DetailsOdyssey Blocking Buffer

Primary Anitbody

Dilution Ratio1:1000 in TBST, overnight, +4 degree celsius

Secondary Antibody

Secondary DescriptionIRDye® 800CW Goat anti-Rabbit
Secondary Manufacturer Cat#926-32211
Secondary Concentration1:10000

Details

Detection NotesOdyssey® CLx Infrared Imaging System
Immunohistochemistry-Paraffin HIF-1 alpha NB100-134
Enlarge
reviewed by:
Liza Sousa
IHC-P Other 01/30/2014
View

Summary

ApplicationImmunohistochemistry-Paraffin
Sample TestedCanine corpus luteum
SpeciesOther

Blocking

Blocking Details0.3% hydrogen peroxide in methanol for 30 min and 10% horse serum in IHC buffer for 20 min

Primary Anitbody

Dilution Ratio1:400, overnight at 4 Celsius degrees

Secondary Antibody

Secondary DescriptionSecondary biotinylated anti-rabbit, anti-mouse, anti-goat IgG antibody
Secondary Manufacturer Cat#Dako, K0690

Details

Detection NotesSubstrate-chromogen solution for 2 min at room temperature
Fixation Details24 h in 4% in buffered formalin, 10 nM citrate buffer pH 6 5 min at room temperature and 15 min in microwave irradiation
Wash DescriptionIHC buffer/0.3% Triton X pH 7.2–7.4 (0.8 mM Na2HPO4, 1.47 mM KH2PO4, 2.68 mM KCl, 137 mM NaCl), 3 x 5 min each wash
Western Blot HIF-1 alpha NB100-134
Enlarge
reviewed by:
Sandhya Khurana
WB Rat 02/05/2012
View

Summary

ApplicationWestern Blot
Sample TestedPC12 nuclear extract, Sample Amount: 50ug
SpeciesRat
CommentsShowed increased in signal intensity of HIF1alpha in cells treated with Cobalt as expected. Also had lysates from cells overexpressing HIF1a (plasmid transfection) as a positive control.

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: 1min, Pos Cnt’l: Lysate from cells overexpressing HIF1alpha, Neg Cnt’l: Untreated cells, Specific Band:110kDa

Comments

CommentsShowed increased in signal intensity of HIF1alpha in cells treated with Cobalt as expected. Also had lysates from cells overexpressing HIF1a (plasmid transfection) as a positive control.
Immunoprecipitation HIF-1 alpha NB100-134
Enlarge
reviewed by:
Kelie Reece
IP Human 10/27/2011
View

Summary

ApplicationImmunoprecipitation
Sample TestedPC-3 Cells
SpeciesHuman
LotK3
CommentsDiluted Lysate Concentration: 1mg/ml, Ab Dilution Ratio: 1 ul, 3 ul, and 5 ul, Protein Capture: Protein A/G Agarose beads

Blocking

Blocking DetailsBlocking Buffer: Odyssey Blocking Buffer, Blocking Time: 1 hour, Blocking Temp: room temperature

Primary Anitbody

Dilution RatioPrimary Ab Dilution Ratio: 1:1000, Primary Ab Incubation Time: overnight, Primary Ab Incubation Temp: 4 degrees Celsius

Secondary Antibody

Secondary DescriptionSecondary Ab: Alexa Fluor 680 goat-anti-mouse IgG, Secondary Ab Dilution: 1:10000

Details

Detection NotesDetection Method: Odyssey imaging system, Negative Control: Normoxia- no cobalt chloride, Specific Bands: 120kDa

Comments

CommentsDiluted Lysate Concentration: 1mg/ml, Ab Dilution Ratio: 1 ul, 3 ul, and 5 ul, Protein Capture: Protein A/G Agarose beads

Product General Protocols

Video Protocols

WB Video Protocol
ICC/IF Video Protocol

FAQs for HIF-1 alpha Antibody (NB100-134). (Showing 1 - 10 of 19 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 19 FAQs.

Control Lysate(s)

Secondary Antibodies

 

Isotype Controls

Other Available Formats

Alexa Fluor 350 NB100-134AF350
Alexa Fluor 405 NB100-134AF405
Alexa Fluor 488 NB100-134AF488
Alexa Fluor 532 NB100-134AF532
Alexa Fluor 594 NB100-134AF594
Alexa Fluor 647 NB100-134AF647
Alexa Fluor 700 NB100-134AF700
Alexa Fluor 750 NB100-134AF750
Biotin NB100-134B
DyLight 350 NB100-134UV
DyLight 405 NB100-134V
DyLight 488 NB100-134G
DyLight 550 NB100-134R
DyLight 594 NB100-134DL594
DyLight 650 NB100-134C
DyLight 680 NB100-134FR
DyLight 755 NB100-134IR
FITC NB100-134F
HRP NB100-134H
Janelia Fluor 549 NB100-134JF549
Janelia Fluor 646 NB100-134JF646

Additional HIF-1 alpha Products

Bioinformatics Tool for HIF-1 alpha Antibody (NB100-134)

Discover related pathways, diseases and genes to HIF-1 alpha Antibody (NB100-134). Need help? Read the Bioinformatics Tool Guide for instructions on using this tool.
Visit Tool

Diseases for HIF-1 alpha Antibody (NB100-134)

Discover more about diseases related to HIF-1 alpha Antibody (NB100-134).
 

Pathways for HIF-1 alpha Antibody (NB100-134)

View related products by pathway.

PTMs for HIF-1 alpha Antibody (NB100-134)

Learn more about PTMs related to HIF-1 alpha Antibody (NB100-134).
 

Research Areas for HIF-1 alpha Antibody (NB100-134)

Find related products by research area.

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.
Coronavirus Brochure

Customers Who Bought This Also Bought

Contact Information

Learn the difference between western blot and simple western

Product PDFs

Calculators

Concentration Calculator

The concentration calculator allows you to quickly calculate the volume, mass or concentration of your vial. Simply enter your mass, volume, or concentration values for your reagent and the calculator will determine the rest.

=
÷

Recent Reviews

4.8
5
11
4
3
3
0
2
0
1
0

KARTHIKEYAN THIRUGNANAM
02/07/2020
Application: WB
Species: Zebrafish

Anonymous
10/28/2018
Application: WB
Species: Human

Anonymous
04/30/2018
Application: WB
Species: Human

Bioinformatics

Gene Symbol HIF1A
Entrez
Uniprot