HIF-1 alpha Antibody

Images

 
Genetic Strategies: Knockdown Validated: HIF-1 alpha Antibody [NB100-449] - Morphological alterations induced by DMOG are HIF-1alpha dependent. glEND.2 cells were stably transfected with shGFP, shHIF-1alpha or ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-449] - HIF-1 alpha induction on Caki-1 cell lysate using CoCl2. Image from verified customer review.
Simple Western: HIF-1 alpha Antibody [NB100-449] - Simple Western lane view shows a specific band for HIF-1 alpha in 0.5 mg/ml of hypoxic HeLa lysate. This experiment was performed under reducing conditions using the ...read more
Immunohistochemistry: HIF-1 alpha Antibody [NB100-449] - Mouse Brain, Neurons 40X
Immunohistochemistry: HIF-1 alpha Antibody [NB100-449] - Detection of human HIF1-alpha by immunohistochemistry. Sample: FFPE section of renal cell carcinoma. Antibody: Affinity purified rabbit anti-HIF1-alpha antibody ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-449] - Detection of Human HIF-1 alpha by Western Blot and Immunoprecipitation. Samples: Whole cell lysate (5, 15 and 50 ug for WB; 1 mg for IP, ...read more
Western Blot: HIF-1 alpha Antibody [NB100-449] - 3T3-L1 mouse embryonic fibroblast adipose-like cell line. Antibody at 1:2000. WB image submitted by a verified customer review.
Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody [NB100-449] - Murine primary bone marrow derived macrophages stained with HIF1-alpha antibody (red). Nuclei were counterstained with DAPI (blue). Image from ...read more
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-449] - Analysis of microvascular density and HIF-1alpha activity. Low, homogenous expression of HIF-1alpha was detected in the tumors of NNK treated mice (40X, ...read more
Flow Cytometry: HIF-1 alpha Antibody [NB100-449] - HeLa cells were treated for 15 hrs with 200 uM CoCl2, fixed in PFA, and permeabilized in 90% MeOH. 1 X 10^6 cells were stained with 0.125 ug anti-HIF-1 alpha and ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-449] - Analysis of HIF-1 alpha in human myeloma cell lysate using anti-HIF-1 alpha. Cells were untreated or treated with IGF-1, IL-6 or CoCl2. ...read more
Western Blot: HIF-1 alpha Antibody [NB100-449] - Detection of HIF-1 alpha in a hypoxic sample. Lane 1: CoCl2 treated Cos-7 nuclear extract (hypoxic). Lane 2: Untreated Cos-7 nuclear extract (normoxic).
Western Blot: HIF-1 alpha Antibody [NB100-449] - Detection of mouse HIF-1 alpha on hypoxia treated MEFs
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-449] - Samples: Whole cell lysate (5, 15 and 50 ug) from HeLa cells that were treated with cobalt chloride (+; 200 uM) or mock treated (-). ...read more
Western Blot: HIF-1 alpha Antibody [NB100-449] - BMDM were seeded at 0.5x10^6 overnight. Cells were treated with 10 ng/ml LPS for 24 hrs. Image from verified customer review.
Western Blot: HIF-1 alpha Antibody [NB100-449] - Analysis of total protein using anti-HIF-1 alpha, -CAIX, -ISCU antibodies and VDACs antibody (PMID: 29596470).
Western Blot: HIF-1 alpha Antibody [NB100-449] - Analysis of total protein using anti-HIF-1 alpha, -ISCU, -FXN antibodies. CoCl2 was used to treat or not HeLa cells for 2 days (PMID: 29596470).
Western Blot: HIF-1 alpha Antibody [NB100-449] - Detection of human HIF1 alpha by western blot. Samples: Whole cell lysate (15 and 50 ug) from HEK293T cells that were either treated with 200 uM cobalt chloride (+) or ...read more
Biological Strategies: Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody [NB100-449] - Formaldehyde-fixed asynchronous HeLa cells.
Immunoprecipitation: HIF-1 alpha Antibody [NB100-449] - HIF-1 alpha analysis in HEK293 cells. Image from verified customer review.
Immunoprecipitation: HIF-1 alpha Antibody [NB100-449] - Detection of human HIF1 alpha by western blot of immunoprecipitates. Samples: Whole cell lysate (1 mg for IP, 20% of IP loaded) from HEK293T cells that were ...read more

Product Details

Summary
Reactivity Hu, Mu, Rt, Ca, Ch, Gt, Pm, Pm, BvSpecies Glossary
Applications WB, Simple Western, ChIP, ELISA, Flow, Flow-IC, ICC/IF, IHC, IHC-Fr, IHC-P, IP, KD, KO
Clonality
Polyclonal
Host
Rabbit
Conjugate
Unconjugated
Concentration
0.2 mg/ml

Order Details

HIF-1 alpha Antibody Summary

Immunogen
The immunogen recognized by this HIF-1 alpha Antibody maps to a region between residues 775 and the C-terminus (residue 826) of human hypoxia-inducible factor 1 [Uniprot# Q16665].
Predicted Species
Bovine (100%). Backed by our 100% Guarantee.
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 1:2000 - 1:10000
  • Simple Western 1:200
  • Chromatin Immunoprecipitation 1:10 - 1:500
  • ELISA 1:100-1:2000
  • Flow Cytometry 0.125 ug per 1 million cells in a 150 mcl reaction
  • Immunocytochemistry/Immunofluorescence 1:10-1:500
  • Immunohistochemistry 1:100 - 1:500
  • Immunohistochemistry-Frozen 1:50-1:200
  • Immunohistochemistry-Paraffin 1:50-1:200
  • Immunoprecipitation 2-5 ug/mg lysate
  • Flow (Intracellular)
  • Knockdown Validated
  • Knockout Validated
Application Notes
ChIP usage was reported in scientific literature (PMID: 25557133). In Simple Western only 10 - 15 uL of the recommended dilution is used per data point. For IHC-P, Tris-EDTA pH 9.0 buffer is recommended for the heat induced epitope retrieval. ELISA (PMID: 17556599 and 16966370). Knockout data (PMID: 31793879). Use in Flow-intracellular reported in scientific literature (PMID:31722203).
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 Brain Cerebral Cortex Whole Tissue Lysate (Adult Whole Normal)
Human Pancreas Whole Tissue Lysate (Adult Whole Tumor)
COS-7 Nuclear Hypoxic Induced Cell Lysate
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 28 Reviews rated 4.3
using
NB100-449 in the following applications:

Publications
Read Publications using
NB100-449 in the following applications:

Reactivity Notes

Monkey (COS-7) and Rat reactivities were reported in customer review feedback. Detection of HIF1 alpha in both Mouse and Human tissue by IHC. Chicken reactivity was reported in scientific literature (PMID: 25632022). Canine reactivity reported in scientific literature (PMID: 28701694). Reactivity with Goat is reported in PMID: 21599540.

Packaging, Storage & Formulations

Storage
Store at 4C. Do not freeze.
Buffer
TBS, 0.1% BSA
Preservative
0.09% Sodium Azide
Concentration
0.2 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 (NB100-449)(231)

We have publications tested in 6 confirmed species: Human, Mouse, Rat, Canine, Chicken, Primate.

We have publications tested in 10 applications: ChIP, ELISA, Flow-IC, ICC/IF, IHC, IHC-Fr, IHC-P, IP, KO, WB.


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ChIP
(3)
ELISA
(2)
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(1)
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(9)
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(16)
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(6)
IHC-P
(12)
IP
(3)
KO
(1)
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(120)
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Human
(57)
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(93)
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(4)
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Showing Publications 1 - 10 of 231. Show All 231 Publications.
Publications using NB100-449 Applications Species
Ghosh P, Guo Y, Ashrafi A et al. Oxygen-enhanced optoacoustic tomography reveals the effectiveness of targeting heme and oxidative phosphorylation at normalizing tumor vascular oxygenation Cancer Res. Jun 16 2020 [PMID: 32546631] (IHC, Human) IHC Human
Hu L, Chen X, Qiu S et al. Intra-Pancreatic Insulin Nourishes Cancer Cells: Do Insulin-Receptor Antagonists such as PGG and EGCG Play a Role? Am. J. Chin. Med. May 28 2020 [PMID: 32468825]
Rawat S Maturation of Cardiac Energy Metabolism in the Normal and Hypertrophied Newborn Heart Thesis Jan 1 2019 (WB, Human) WB Human
Kim KY, Kim YR, Choi KW et al. Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients Transl Neurodegener Jul 1 2020 [PMID: 32605607]
Leinhos L Myofibroblast differentiation in hypoxia: a novel role for ArhGAP29 Thesis (WB) WB
van Dam TP DNA polymerase beta prevents AID-instigated mutagenic non-canonical mismatch DNA repair bioRxiv Jan 1 2020 (IP, Mouse) IP Mouse
Sil S, Hu G, Liao K et al. HIV-1 Tat-mediated astrocytic amyloidosis involves the HIF-1 alpha/lncRNA BACE1-AS axis PLoS Biol. May 26 2020 [PMID: 32453744] (ICC/IF, Human) ICC/IF Human
Doigneaux C, Pedley AM, Mistry IN et al. Hypoxia Drives the Assembly of the Multi-Enzyme Purinosome Complex J. Biol. Chem. May 21 2020 [PMID: 32439803] (WB, Human) WB Human
Sola-Penna M, PaixAo LP, Branco JR, et al. Serotonin activates glycolysis and mitochondria biogenesis in human breast cancer cells through activation of the Jak1/STAT3/ERK1/2 and adenylate cyclase/PKA, respectively Br. J. Cancer Dec 10 2019 [PMID: 31819176] (WB, Human) WB Human
Di Gioia M, Spreafico R, Springstead JR et al. Endogenous oxidized phospholipids reprogram cellular metabolism and boost hyperinflammation Nat. Immunol. Nov 25 2019 [PMID: 31768073] (WB, Mouse) WB Mouse
Show All 231 Publications.

Reviews for HIF-1 alpha Antibody (NB100-449) (28) 4.328

Average Rating: 4.3
(Based on 28 reviews)
We have 28 reviews tested in 5 species: Human, Mouse, Rat, Goat, Other.

Reviews using NB100-449:
Filter by Applications
WB
(23)
IHC-P
(1)
IP
(2)
IF
(1)
Simple Western
(1)
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Filter by Species
Human
(10)
Mouse
(10)
Rat
(2)
Goat
(1)
Other
(3)
All Species
Images Ratings Applications Species Date Details
Western Blot HIF-1 alpha NB100-449
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reviewed by:
Anonymous
WB Mouse 05/08/2020
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Summary

ApplicationWestern Blot
Sample Tested3T3-L1 mouse embryonic fibroblast adipose-like cell line
SpeciesMouse
Lotlot A3

Comments

Commentsskimmed milk 5%
primary : 1:2000
secondary: 1: 200000
Immunohistochemistry-Paraffin HIF-1 alpha NB100-449
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reviewed by:
Anonymous
IHC-P Mouse 06/07/2019
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Summary

ApplicationImmunohistochemistry-Paraffin
Sample TestedPDX mouse
SpeciesMouse
LotLot A3

Comments

CommentsWorking Dilution 1:100, EDTA pH 9.0; detection kit Polymer-HRP.
Western Blot HIF-1 alpha NB100-449
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reviewed by:
an shiyu
WB Goat 05/15/2018
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Summary

ApplicationWestern Blot
Sample Testedlaydig cell
SpeciesGoat
LotNBP1-04676SS

Comments

Commentsdilution:1:500
Western Blot HIF-1 alpha NB100-449
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reviewed by:
Anonymous
WB Mouse 07/17/2017
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Summary

ApplicationWestern Blot
Sample TestedBMDM
SpeciesMouse
LotA5
 
reviewed by:
Anonymous
IP Rat 04/24/2017
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Summary

ApplicationImmunoprecipitation
Sample TestedPC-12 rat adrenal pheochromocytoma cell line
SpeciesRat
LotA6
Immunofluorescence HIF-1 alpha NB100-449
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reviewed by:
Anonymous
IF Mouse 01/12/2016
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Summary

ApplicationImmunofluorescence
Sample Testedprimary bone marrow derived macrophages
SpeciesMouse

Blocking

Blocking Details-

Details

Detection Notes-

Comments

CommentsMurine primary bone marrow derived macrophages probed with rabbit HIF1a antibody (1:100) in blocking buffer at 4°C overnight, secondary goat anti-rabbit AF647 (1:250) for 2 h at ambient temperature, Cells were mounted with Vectashield containing DAPI for imaging. Image (top right) derived from merge of DAPI and AF647 staining. White arrows point at cells containing nuclear HIF1a. Unstained and secondary antibody controls shown in bottom panels, Magnification, 63X
 
reviewed by:
Anonymous
WB Human 09/07/2015
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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 Concentration1:2000

Details

Detection NotesRegular Protocol
Simple Western HIF-1 alpha NB100-449
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reviewed by:
Zachary Patinkin
Simple Western Human 06/09/2015
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Summary

ApplicationSimple Western
Sample TestedHuman mesenchymal stem cell whole cell lysate
SpeciesHuman
LotA5

Blocking

Blocking DetailsProtein Simple Antibody Diluent, 5 min, 25C

Primary Anitbody

Dilution Ratio1:200, 30 minutes, Protein Simple Antibody Diluent

Secondary Antibody

Secondary DescriptionProtein Simple Anti-Rabbit Secondary
Secondary Manufacturer Cat#042-206
Secondary Concentrationundiluted

Details

Detection NotesWES multi-image analysis
Western Blot HIF-1 alpha NB100-449
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reviewed by:
Enrica Borsi
WB Human 01/09/2015
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Summary

ApplicationWestern Blot
Sample TestedMyeloma cells whole cell lysate
SpeciesHuman

Blocking

Blocking DetailsBlocking buffer Milk 5%

Details

Detection NotesWestern blot
 
reviewed by:
Anonymous
WB Human 12/12/2014
View

Summary

ApplicationWestern Blot
Sample TestedSee PMID 22923663
SpeciesHuman

Blocking

Blocking DetailsSee PMID 22923663

Details

Detection NotesSee PMID 22923663

Comments

CommentsPublished in PMID: 22923663
Western Blot HIF-1 alpha NB100-449
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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 lysates
SpeciesHuman
LotA4
CommentsAlthough I got my result as expected, this antibody has weak bands and dirty background in my western blotting.

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 Notes4 min exposure shows band at correct MW.

Comments

CommentsAlthough I got my result as expected, this antibody has weak bands and dirty background in my western blotting.
Western Blot HIF-1 alpha NB100-449
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reviewed by:
Xiuquan Luo
WB Human 07/18/2014
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Summary

ApplicationWestern Blot
Sample TestedCaki-1 cell lysate
SpeciesHuman

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

Details

Detection NotesECL, exposured for 1 min
Western Blot HIF-1 alpha NB100-449
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reviewed by:
Anonymous
WB Mouse 07/05/2014
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Summary

ApplicationWestern Blot
Sample Testedlarge intestinal lamina propria leukocytes (LPLs).
SpeciesMouse
 
reviewed by:
Paramananda Saikia
WB Mouse 06/30/2014
View

Summary

ApplicationWestern Blot
Sample TestedMouse
SpeciesMouse
LotA4

Blocking

Blocking Details5% bsa

Primary Anitbody

Dilution Ratio1:1500

Secondary Antibody

Secondary Descriptiongoat anti-rabbit
Western Blot HIF-1 alpha NB100-449
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reviewed by:
Alshaimaa Adawy
WB Human 04/29/2014
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Summary

ApplicationWestern Blot
Sample Testednuclear fractions from T47-D beast cancer cell line
SpeciesHuman
LotA5

Blocking

Blocking Detailsblocking: 5% BSA for 1 hour at room temprature

Primary Anitbody

Dilution Ratio1:1000, overnight at 4 C in BSA

Secondary Antibody

Secondary DescriptionAnti-rabbit IgG, HRP-linked Antibody
Secondary Manufacturer Cat#7074s, cell signaling
Secondary Concentration1:5000

Details

Detection NotesVilber Lourmat imager, using home made ECL, exposure time 3 minutes
Western Blot HIF-1 alpha NB100-449
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reviewed by:
Anonymous
WB Other 03/14/2014
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Summary

ApplicationWestern Blot
SpeciesOther
LotA4
Pub Med ID21193390
FileView PDF
Western Blot HIF-1 alpha NB100-449
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reviewed by:
Lidan Zhao
WB Other 08/26/2013
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Summary

ApplicationWestern Blot
Sample Testedpig satellite cell nuclear protein
SpeciesOther
CommentsI first ordered NB100-105 and tried several times, but it didn't work with my samples. I contacted technical support and they sent me a free vial of NB100-449 as a replacement. I found that this antibody worked well both on the positive control and my pig samples.

Blocking

Blocking Details5% milk in TBST

Primary Anitbody

Dilution Ratio1:500 in 5% milk, 4 C overnight

Secondary Antibody

Secondary DescriptionIRDye 800CW Goat anti-Rabbit IgG
Secondary Concentration1:20000

Details

Detection NotesIRDye conjugated secondary scanned by Licor Odyssey system. Used Novus COS-7 nuclear extracts (NB800-PC26) as positive control

Comments

CommentsI first ordered NB100-105 and tried several times, but it didn't work with my samples. I contacted technical support and they sent me a free vial of NB100-449 as a replacement. I found that this antibody worked well both on the positive control and my pig samples.
 
reviewed by:
Anonymous
WB Human 11/05/2012
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Summary

ApplicationWestern Blot
Sample Testedhuman cell lysates
SpeciesHuman

Blocking

Blocking Details5% Non fat milk

Primary Anitbody

Dilution Ratio1:500

Secondary Antibody

Secondary Descriptionanti-rabbit-HRP
Secondary Manufacturer Cat#Sigma
Secondary Concentration1:1000

Details

Detection NotesECL
Western Blot HIF-1 alpha NB100-449
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reviewed by:
Annie Curtis
WB Mouse 08/24/2012
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Summary

ApplicationWestern Blot
Sample Testedmouse peritoneal and bone marrow derived macrophages
SpeciesMouse
LotA4
CommentsWe tried numerous HIF1alpha antibodies with little success, we finally got this one to work but one thing that is necessary is that you need to lyse your cells directly in loading buffer. We used about 100uL of Laemmli buffer per 6 well dish, then boil immediately after lysing. Samples can then be either loaded directly onto the gel or stored at -20. However, we did find that if samples were stored for too long at -20 then we did lose the ability to detect the protein.

Blocking

Blocking Detailsblocked in 5% milk for 1 hour at room temperature

Primary Anitbody

Dilution Ratio1:1000 overnight at 4deg C, it has to be an overnight incubation to work

Secondary Antibody

Secondary DescriptionGoat anti-rabbit IgG, HRP
Secondary Concentration1: 1000

Details

Detection NotesECL exposure of 2 minutes needed

Comments

CommentsWe tried numerous HIF1alpha antibodies with little success, we finally got this one to work but one thing that is necessary is that you need to lyse your cells directly in loading buffer. We used about 100uL of Laemmli buffer per 6 well dish, then boil immediately after lysing. Samples can then be either loaded directly onto the gel or stored at -20. However, we did find that if samples were stored for too long at -20 then we did lose the ability to detect the protein.
Western Blot HIF-1 alpha NB100-449
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reviewed by:
kimberley tam
WB 08/07/2012
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Summary

ApplicationWestern Blot
LotA2
Special ApplicationsExcellent antibody. Tested with mouse
Pub Med ID20600586
 
reviewed by:
Anonymous
WB 05/09/2012
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Summary

ApplicationWestern Blot
Pub Med ID16611993
FileView PDF
 
reviewed by:
Anonymous
WB Rat 01/03/2012
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Summary

ApplicationWestern Blot
Sample TestedPC12 cell whole lysate, Sample Amount: 25ug
SpeciesRat
LotA4
CommentsThis antibody could detect HIF-1a but weak.

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

Secondary Antibody

Secondary DescriptionSecondary Ab: anti-Rabbit IgG, Secondary Ab Dilution Ratio: 1:5000

Details

Detection NotesDetection Method: ECL Plus

Comments

CommentsThis antibody could detect HIF-1a but weak.
Western Blot HIF-1 alpha NB100-449
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reviewed by:
Anonymous
WB Human 06/08/2011
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Summary

ApplicationWestern Blot
Sample TestedHUVEC whole cell lysate, Sample Amount: 20 microg
SpeciesHuman
CommentsWestern blot analysis showing that the increase in HIF-1alpha levels triggered by hypoxia in HUVEC is prevented by a specific siRNA against HIF-1alpha (siHIFa).(N: Normoxia, H: Hypoxia; siRAND: Random siRNA
Pub Med ID21622104
FileView PDF

Blocking

Blocking DetailsBlocking Buffer: 5%Milk in TBST, Blocking Time: overnight, Blocking Temp: 4 degrees Celsius

Primary Anitbody

Dilution RatioDilution Ratio: 1:500, Incubation Dilution Buffer: 5% Milk in TBST, Incubation Time: 1 hour, Incubation Temp: room temperature

Secondary Antibody

Secondary DescriptionSecondary Ab: anti-rabbit DAKO, Secondary Ab Dilution Ratio: 1:2000
Secondary Manufacturer Cat#P0448

Details

Detection NotesMethod: SuperSignal West, Time: 3min, Controls: Hypoxia/normoxia in random transfected cells, Bands: Multiband at 110kDa

Comments

CommentsWestern blot analysis showing that the increase in HIF-1alpha levels triggered by hypoxia in HUVEC is prevented by a specific siRNA against HIF-1alpha (siHIFa).(N: Normoxia, H: Hypoxia; siRAND: Random siRNA
Western Blot HIF-1 alpha NB100-449
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reviewed by:
Anonymous
WB Mouse 12/22/2010
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Summary

ApplicationWestern Blot
Sample Testedcerebellar neurons
SpeciesMouse
CommentsThis Hif1 alpha antibody is the only one we have found to work consistently well. We use it 1:500 in 1% BSA and reuse it for 4-5 times. We get a a doublet, the expected band at around 100KDa and another band at about 130KDa or so (right below the 150KDa marker.)

Blocking

Blocking DetailsBlocking Buffer: 3% BSA in TBST, Blocking Time: 1 hour, Blocking Temp: Room temperature

Primary Anitbody

Dilution RatioPrimary Ab Dilution Ratio: 1:500, Primary Ab Incubation Time: overnight, Primary Ab Incubation Temp: 4 degrees Celsius

Secondary Antibody

Secondary DescriptionSecondary Ab Dilution Ratio: 1:5000

Details

Detection NotesDetection Method: ECL, Exposure Time: 5 minutes

Comments

CommentsThis Hif1 alpha antibody is the only one we have found to work consistently well. We use it 1:500 in 1% BSA and reuse it for 4-5 times. We get a a doublet, the expected band at around 100KDa and another band at about 130KDa or so (right below the 150KDa marker.)
Immunoprecipitation HIF-1 alpha NB100-449
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reviewed by:
William Lin
IP Human 12/03/2010
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Summary

ApplicationImmunoprecipitation
Sample TestedHEK 293
SpeciesHuman
LotA3
CommentsI used BD mouse anti-human HIF1 antibody for WB because I didn't want to detect the rabbit IgG from the NB100-449 antibody during the IP. Lysate Amount: 1mg, Lysate Preclear: agarose A, Diluted Lysate Concentration: 2ug/ul, Ab Dilution Ratio: 1:100, Protein Capture: agarose A, Collection Method: 4000 rpm centrifugation, Protein Reduction: 2X sample buffer, with SDS/bME, Bead Removal: 4min, 4C

Blocking

Blocking DetailsBlocking Buffer: 5% milk in TBST, Blocking Time: 2 hours, Blocking Temp: 4 degrees Celsius

Primary Anitbody

Dilution RatioIncubation Dilution Buffer: 5% milk/TBST, Dilution Ratio: 1:2000, Incubation Time: overnight, Incubation Temp: 4 C

Secondary Antibody

Secondary DescriptionSecondary Ab: anti-human IgG-HRP, Secondary Ab Dilution: 1:10000
Secondary Manufacturer Cat#promega

Details

Detection NotesMethod: ECL, Exposure Time: 2 min, Pos.Ctrl: DFO treated, Neg.Ctrl: no treatment, Specific Bands: 116kDa

Comments

CommentsI used BD mouse anti-human HIF1 antibody for WB because I didn't want to detect the rabbit IgG from the NB100-449 antibody during the IP. Lysate Amount: 1mg, Lysate Preclear: agarose A, Diluted Lysate Concentration: 2ug/ul, Ab Dilution Ratio: 1:100, Protein Capture: agarose A, Collection Method: 4000 rpm centrifugation, Protein Reduction: 2X sample buffer, with SDS/bME, Bead Removal: 4min, 4C
 
reviewed by:
Anonymous
WB Other 03/11/2010
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Summary

ApplicationWestern Blot
Sample TestedColon Cancer Cell lines, Sample Amount: 50ug
SpeciesOther
LotA2
CommentsThe lanes on the left are HCT116 treated with increasing concentrations of DMOG. The lanes on the right are HCT116 Hif1-/- cells with the same treatment.

Blocking

Blocking DetailsBlocking Buffer: 5% Blotto, Blocking Time: 1 hour, Blocking Temp: Room temperature

Primary Anitbody

Dilution RatioDilution Ratio: 1/5000, Incubation Dilution Buffer: 5% BSA in TNS with Tween, Incubation Time: overnight, Incubation Temp: 4C

Secondary Antibody

Secondary DescriptionSecondary Ab: Cell Signaling Goat Anti-Rabbit HRP, Secondary Ab Dilution Ratio: 1/10000

Details

Detection NotesDetection Method: ECL Plus, Specific Bands: 120 kDa

Comments

CommentsThe lanes on the left are HCT116 treated with increasing concentrations of DMOG. The lanes on the right are HCT116 Hif1-/- cells with the same treatment.
 
reviewed by:
Anonymous
WB Mouse 12/21/2009
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Summary

ApplicationWestern Blot
Sample Testedhuman tumor model mouse, Sample Amount: 12ug
SpeciesMouse
CommentsNuclear protein extracts: Chen et al. J Cell Biochem 2001;82(3):512-521.

Blocking

Blocking DetailsBlocking Buffer: 5% BSA, Blocking Time: 1 hour, Blocking Temp: Room temperature

Primary Anitbody

Dilution RatioDilution Ratio: 1/500, Incubation Dilution Buffer: TBST with 5% milk, Incubation Time: overnight, Incubation Temp: 4 C

Secondary Antibody

Secondary DescriptionSecondary Ab: Anti-rabbit IgG HRP-linked, Cell Signaling #7047, Secondary Ab Dilution Ratio: 1/3000

Details

Detection NotesDetection Method: ECL, Exposure Time: 5 - 10 min., Specific Bands: 100 kDa

Comments

CommentsNuclear protein extracts: Chen et al. J Cell Biochem 2001;82(3):512-521.
 
reviewed by:
Anonymous
WB Mouse 04/08/2009
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Summary

ApplicationWestern Blot
Sample TestedMouse Neuroblastoma, Sample Amount: 25ug
SpeciesMouse
LotA2
Comments"Load:
Empty Vector Normoxia
Empty Vector Hypoxia</p><p>Hif1a over expression Normoxia
Hif1a over expression Hypoxia"

Blocking

Blocking DetailsBlocking Buffer: 5% NFDM with TBS and.5% Tween 20, Blocking Time: 90 minutes, Blocking Temp: 25 degrees Celsius

Primary Anitbody

Dilution RatioDilution Ratio: 1/500, Incubation Dilution Buffer: 5% NFDM w/ TBS and.5% Tween 20, Incubation Time/Temp: overnight(16hrs) at 4C

Secondary Antibody

Secondary DescriptionSecondary Ab: Santa Cruz Goat anti Rabbit Secondary, Secondary Ab Dilution Ratio: 1/5000

Details

Detection NotesMethod: Chemiluminescence onto Kodak XAR film, Exposure Time: 2-15 min, Bands: 120,60 kDa(specific), 50 kDa(non-specific)

Comments

Comments"Load:
Empty Vector Normoxia
Empty Vector Hypoxia</p><p>Hif1a over expression Normoxia
Hif1a over expression Hypoxia"

Product General Protocols

Video Protocols

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

FAQs for HIF-1 alpha Antibody (NB100-449). (Showing 1 - 10 of 14 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 14 FAQs.

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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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05/08/2020
Application: WB
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Bioinformatics

Gene Symbol HIF1A
Entrez
Uniprot