HIF-1 alpha Antibody (H1alpha67)

Images

 
Biological Strategies: Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF-1 alpha induction by CoCl2 on Caki-1 cell lysate. WB image submitted by a verified customer review.
Genetic Strategies: Knockout Validated: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF-1 alpha was detected in immersion fixed DFO treated Hela cells (left) but was not detected in HIF-1 knockout HeLa cells ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Naive CD4 T cells from WT, VHL-deficient (Vhl KO), or HIF-1 alpha-deficient (HIF-1 alpha KO) mice were differentiated under ...read more
Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Staining of HIF-1 alpha in human kidney using NB100-105. Renal tubular epithelium showed moderate membranous, cytoplasmic and nuclear staining, and ...read more
Flow Cytometry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Analysis using the Alexa Fluor (R) 488 conjugate of NB100-105. Staining of HIF-1 alpha in multiple myeloma cells: H929 cells (0.5 x 10^6) were stained with ...read more
Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF1alpha/PFKFB3 is upregulated in beta-cells of HIP rats and humans with type 2 diabetes. Representative Western blot of PFKFB3 and HIF1alpha levels in ...read more
Biological Strategies: Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF-1 alpha was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line ...read more
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Analysis using the biotin conjugate of NB100-105. Staining of human glioblastoma multiforme.
Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Upregulation of HIF-1alpha in human PCa. Protein expression of HIF-1alpha, VEGF, and GLUT4 were examined with western blot, in PC-3, DU145, and LNCaP cells ...read more
Immunohistochemistry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - HIF-1alpha expression in PDAC and adjacent normal pancreatic tissue. PDAC with weak (intensity 1) nuclear and cytoplasmic HIF-1alpha staining (left). ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Analysis of HIF-1 alpha in human hepatocytes from cancer patient using anti-HIF-1 alpha antibody NB100-105. Western blot image ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Analysis using the HRP conjugate of NB100-105. Detection of 50 ug cobalt chloride induced COS-7 nuclear extracts (NB800-PC26) ...read more
Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - L. donovani infection induces HIF-1 alpha expression in CD11chi splenic DCs in an IRF-5 dependent manner. Mice were infected with 2 x 10^7 amastigotes ...read more
Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Upregulation of HIF-1alpha in human Pca. HIF-1alpha protein was detected by western blot in nonmalignant (RWPE-1 and BPH1) and PCa cell lines (PC-3, DU145, ...read more
Flow Cytometry: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Analysis using the Alexa Fluor(R) 488 conjugate of NB100-105. Staining of HIF-1 alpha in H929 cells using HIF-1 alpha antibody. Flow cytometry image ...read more

Product Details

Summary
Reactivity Hu, Mu, Rt, Po, Bv, Ca, Fe, Ma, Pm, Pm, Rb, Sh, XpSpecies Glossary
Applications WB, ChIP, ELISA, Flow, GS, IA, IB, ICC/IF, IHC, IHC-Fr, IHC-P, In vitro, IP, LA, PLA, TCS, KO
Clone
H1alpha67
Clonality
Monoclonal
Host
Mouse
Conjugate
Unconjugated
Concentration
1.0 mg/ml

Order Details

HIF-1 alpha Antibody (H1alpha67) Summary

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

Applications/Dilutions

Dilutions
  • Western Blot 1:500
  • Chromatin Immunoprecipitation 1 - 5 ug/IP
  • ELISA 1:100 - 1:2000
  • Flow Cytometry 1:10 - 1:1000
  • Gel Super Shift Assays 1:1 - 1:100
  • Immunoassay
  • Immunoblotting
  • Immunocytochemistry/Immunofluorescence 1:50
  • Immunohistochemistry 1:20 - 1:50
  • Immunohistochemistry-Frozen 1:20 - 1:50
  • Immunohistochemistry-Paraffin 1:20 - 1:50
  • Immunoprecipitation 1:10 - 1:500
  • In vitro assay
  • Proximity Ligation Assay
  • Tissue Culture Substratum
  • Knockout Validated
  • Ligand Activation
Application Notes
ChIP usage was reported in scientific literature. ELISA usage was reported in scientific literature (PMID: 20042684). Gel Super Shift Assays usage was reported in scientific literature (PMID: 22411794). Ligand Activation usage was reported in scientific literature (PMID: 26147748). Use in Immunoassay reported in scientific literature (PMID: 26147748). Use in vitro assay reported in multiple pieces of scientific literature. In WB, a band can be seen at 120 kDa representing HIF-1 alpha in induced tissues and cells. Multiple bands may be seen at 100-120 kDa representing post-translational modification of HIF-1 alpha. For WB, testing on nuclear extracts is recommended. Knock Out Validation was reported in scientific literature (PMID: 26861754). Use in proximity ligation assay reported in scientific literature (PMID 27595394). Use in Immunoblotting reported in multiple pieces of scientific literature. We recommend the use of a highly sensitive ECL reagent, such as West Pico PLUS, for Western blot detection.
Theoretical MW
93 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Control
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 40 Reviews rated 4.2
using
NB100-105 in the following applications:

Publications
Read Publications using
NB100-105 in the following applications:

  • 49 publications
  • 6 publications
  • 3 publications
  • 1 publication
  • GS
    7 publications
  • IA
    1 publication
  • IB
    1 publication
  • 56 publications
  • IF
    1 publication
  • IHC
    124 publications
  • 6 publications
  • 94 publications
  • 1 publication
  • IP
    32 publications
  • KD
    1 publication
  • PLA
    2 publications
  • WB
    445 publications

Reactivity Notes

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

Packaging, Storage & Formulations

Storage
Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
Buffer
PBS, 1% BSA
Preservative
0.05% Sodium Azide
Concentration
1.0 mg/ml
Purity
Protein G purified

Alternate Names for HIF-1 alpha Antibody (H1alpha67)

  • 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
  • H1alpha67
  • 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 (NB100-105)(849)

We have publications tested in 10 confirmed species: Human, Mouse, Rat, Bovine, Canine, Porcine, Primate, Rabbit, Sheep, Xenopus.

We have publications tested in 17 applications: ChIP, ELISA, EMSA, FLOW, GS, IA, IB, ICC/IF, IF, IHC, IHC-Fr, IHC-P, IHC/IF, IP, KD, PLA, WB.


Filter By Application
ChIP
(49)
ELISA
(6)
EMSA
(3)
FLOW
(1)
GS
(7)
IA
(1)
IB
(1)
ICC/IF
(56)
IF
(1)
IHC
(124)
IHC-Fr
(6)
IHC-P
(94)
IHC/IF
(1)
IP
(32)
KD
(1)
PLA
(2)
WB
(445)
All Applications
Filter By Species
Human
(386)
Mouse
(229)
Rat
(115)
Bovine
(5)
Canine
(3)
Porcine
(5)
Primate
(2)
Rabbit
(7)
Sheep
(3)
Xenopus
(1)
All Species
Showing Publications 1 - 10 of 849. Show All 849 Publications.
Publications using NB100-105 Applications Species
Kondrashina, A. A Phosphorescent Nanoparticle-Based Probe for Sensing and Imaging of (Intra)Cellular Oxygen in Multiple Detection Modalities Adv Funct Mater 2012 (ICC/IF, Rat) ICC/IF Rat
Hoefflin R, Harlander S, SchAfer S et al. HIF-1 alpha and HIF-2 alpha differently regulate tumour development and inflammation of clear cell renal cell carcinoma in mice Nat Commun Jan 1 2020 [PMID: 32807776] (IHC, Mouse) IHC Mouse
Wang Z, Cui M, Qu Y et al. Hypoxia protects rat bone marrow mesenchymal stem cells against compression-induced apoptosis in degenerative disc microenvironment through activation of the HIF-1 alpha/YAP signaling pathway Stem Cells Dev. Aug 16 2020 [PMID: 32799744] (WB) WB
Fang X, Zhang D, Zhao W et al. Dishevelled Associated Activator Of Morphogenesis (DAAM) Facilitates Invasion of Hepatocellular Carcinoma by Upregulating Hypoxia-Inducible Factor 1 alpha (HIF-1 alpha) Expression Med. Sci. Monit. Aug 9 2020 [PMID: 32772041] (WB, KD) WB, KD
Xiong Z, Ding J, Zhou J et al. Correlation between the HIF-1 alpha/Notch signaling pathway and Modic changes in nucleus pulposus cells isolated from patients with low back pain BMC Musculoskelet Disord Jul 28 2020 [PMID: 32723315] (WB, Human) WB Human
Zhang ZY, Zhang SL, Chen HL et al. The up-regulation of NDRG1 by HIF counteracts the cancer-promoting effect of HIF in VHL-deficient clear cell renal cell carcinoma Cell Prolif. Jun 14 2020 [PMID: 32537867] (IHC, Human) IHC Human
Zong S, Tang Y, Li W et al. A Chinese Herbal Formula Suppresses Colorectal Cancer Migration and Vasculogenic Mimicry Through ROS/HIF-1alpha/MMP2 Pathway in Hypoxic Microenvironment Front. Pharmacol. May 15 2020 [PMID: 32499699]
Ho JJD, Balukoff NC, Theodoridis PR et al. A network of RNA-binding proteins controls translation efficiency to activate anaerobic metabolism Nat Commun May 29 2020 [PMID: 32472050] (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]
Chen YL, Sheu JJ, Sun CK et al. MicroRNA-214 modulates the senescence of vascular smooth muscle cells in carotid artery stenosis Mol. Med. May 14 2020 [PMID: 32410577] (WB, Rat) WB Rat
Show All 849 Publications.

Reviews for HIF-1 alpha Antibody (NB100-105) (40) 4.240

Average Rating: 4.2
(Based on 40 reviews)
We have 40 reviews tested in 5 species: Human, Mouse, Rat, Other, also tested in mouse and rat.

Reviews using NB100-105:
Filter by Applications
WB
(34)
IHC
(1)
IF
(2)
ICC
(2)
All Applications
Filter by Species
Human
(20)
Mouse
(12)
Rat
(4)
Other
(3)
also tested in mouse and rat
(1)
All Species
Images Ratings Applications Species Date Details
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Human 08/09/2020
View

Summary

ApplicationWestern Blot
Sample TestedHEK293A whole cell lysate,RCC4 Whole cell lysate
SpeciesHuman
LotBH-23
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Human 06/13/2019
View

Summary

ApplicationWestern Blot
Sample TestedBlood mononuclear cells (PBMCs)
SpeciesHuman
LotBH-13

Comments

CommentsConfirmation of induction of hypoxia in CoCl2 treated PBMCs infected with vGPCR frame shift knock out and wild type revertant KSHV virion. KSHV infected cells were treated with CoCl2 for 48 hours followed by protein isolation and western blot against HIF1 alpha.
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Human 01/29/2019
View

Summary

ApplicationWestern Blot
Sample Testedhuman adrenal cancer cells (whole cell lysate)
SpeciesHuman
Lotunknown
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Rat 01/29/2019
View

Summary

ApplicationWestern Blot
Sample TestedRat liver
SpeciesRat
LotAW-1

Comments

CommentsDetection Method: ECL
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Human 01/24/2019
View

Summary

ApplicationWestern Blot
Sample Tested Hela whole cell lysate
SpeciesHuman
LotAB7

Comments

CommentsECL solution for 5min, then exposure to film 3-5min
 
reviewed by:
Anonymous
WB Human 01/23/2019
View

Summary

ApplicationWestern Blot
Sample Tested293t cell lysate
SpeciesHuman
LotNB100-105
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Human 06/29/2017
View

Summary

ApplicationWestern Blot
Sample Tested293t cell lysate
SpeciesHuman
Lotbh-1
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Mouse 02/01/2017
View

Summary

ApplicationWestern Blot
Sample TestedCD4 T cells cultured in vitro, stimulated with anti-CD3/anti-CD28
SpeciesMouse

Comments

CommentsFigure Legend: Naïve CD4 T cells from WT, VHL-deficient (Vhl KO), or HIF-1a–deficient (Hif1a KO) mice were differentiated under IL-22–skewing conditions for a total of 60 h. Some cells remained at normoxia for the duration of the culture (N); others were at normoxia for 35 h and then hypoxia (1% O2) for 24 h (H). At 60 h, nuclear extracts were harvested, and HIF-1a and lamin B1 levels were analyzed by Western blot.
 
reviewed by:
Anonymous
also tested in mouse and rat 12/03/2016
View

Summary

Sample Tested2 Sample tested
Speciesalso tested in mouse and rat
LotN/A
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
michael dodd
WB Mouse 08/17/2016
View

Summary

ApplicationWestern Blot
Sample TestedHL-1 cardiomyocytes
SpeciesMouse

Blocking

Blocking Details5% milk, in TBST for 1 hour at room temperature

Primary Anitbody

Dilution Ratio1 in 500, Overnight at 4C, in 5% milk with TBST

Secondary Antibody

Secondary DescriptionGoat anti-mouse, HRP
Secondary Concentration1 in 2000

Details

Detection NotesECL Prime, Licor scanner, 12 min exposure
 
reviewed by:
Anonymous
WB Human 05/08/2016
View

Summary

ApplicationWestern Blot
Sample Testedk562 cells
SpeciesHuman
LotBA-3

Blocking

Blocking Details2hs,milk

Primary Anitbody

Dilution Ratio1/500

Secondary Antibody

Secondary Concentration1/2000

Details

Detection Notes45s
 
reviewed by:
Anonymous
WB Human 04/18/2016
View

Summary

ApplicationWestern Blot
Sample TestedHEK293A whole cell lysate
SpeciesHuman
LotAW-1

Blocking

Blocking Details4% milk in TBST, 1hr at room temperature

Primary Anitbody

Dilution Ratio1:2000 dilution in TBST buffer, incubated at 4 degrees overnight (~16hrs)

Secondary Antibody

Secondary DescriptionGoat anti-mouse IgG
Secondary Manufacturer Cat#ThermoScientific #31430
Secondary Concentration1:7500

Details

Detection NotesBio-Rad Versadoc, exposure time was one image every 15s for 5 minutes, negative control was empty plasmid
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
MENG xing
WB Mouse 03/24/2016
View

Summary

ApplicationWestern Blot
SpeciesMouse
LotBD-10
Special ApplicationsBone marrow cells
FileView PDF
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Mouse 10/07/2015
View

Summary

ApplicationWestern Blot
Sample TestedCell lysate
SpeciesMouse

Blocking

Blocking DetailsMilk

Primary Anitbody

Dilution RatioOvernight 1:500

Secondary Antibody

Secondary Descriptionanti-rabbit

Details

Detection NotesECL (doesn't work on Licor)
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Zhongxiao Wang
WB Mouse 10/05/2015
View

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 band

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 Ratio1:500, overnight

Secondary Antibody

Secondary DescriptionSheep anti-mouse IgG, HRP
Secondary Manufacturer Cat#GE Healthcare, NA931V
Secondary Concentration1:2000

Details

Detection NotesRegular Protocol
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Human 08/10/2015
View

Summary

ApplicationWestern Blot
Sample TestedHuman prostate cancer cell line protein samples
SpeciesHuman

Blocking

Blocking Details5% milk in TBS buffer supplemented with 0.1% tween20 at room temperature for one hour

Primary Anitbody

Dilution Ratio1:1000

Secondary Antibody

Secondary DescriptionAnti-Rabbit secondary diluted at 1:3000 in 5% milk
Secondary Manufacturer Cat#SC-2005
Secondary Concentration0.4 mg/ml

Details

Detection NotesWestern blotting using ECL reagent (purchased from GE life sciences) and x-ray films.
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Human 04/28/2015
View

Summary

ApplicationWestern Blot
SpeciesHuman
Special Applicationshuman hepatocytes from cancer patient
FileView PDF
 
reviewed by:
Anonymous
WB Mouse 12/17/2014
View

Summary

ApplicationWestern Blot
Sample TestedSee PIMD: 23148210
SpeciesMouse

Blocking

Blocking DetailsSee PIMD: 23148210

Details

Detection NotesSee PIMD: 23148210

Comments

CommentsPublished in PMID: 23148210
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Xiuquan Luo
WB Human 07/18/2014
View

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-mouse IgG-HRP
Secondary Manufacturer Cat#sc-2005
Secondary Concentration1:2000 dilution

Details

Detection NotesECL, exposured for 5 min
 
reviewed by:
Anonymous
WB Human 07/01/2014
View

Summary

ApplicationWestern Blot
Sample TestedHela Cell Lysate
SpeciesHuman
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Jessica Marinello
WB Human 05/09/2014
View

Summary

ApplicationWestern Blot
Sample TestedHela whole cell lysate
SpeciesHuman

Blocking

Blocking DetailsTBS milk 5% for 1 hour room temperature

Primary Anitbody

Dilution RatioAntibody diluition 1:1000 in TBS milk 5%, incubation time 2 hours room temperature

Secondary Antibody

Secondary DescriptionGoat anti-mouse IgG, HRP
Secondary Manufacturer Cat#santa cruz

Details

Detection NotesECL plus Western Blotting Detection Kit 5 minutes incubation room temperature, Phosphorimager detection system,
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Mouse 04/28/2014
View

Summary

ApplicationWestern Blot
Sample Testedmouse brain lysate
SpeciesMouse

Blocking

Blocking Details7% Milk 30 min RT

Primary Anitbody

Dilution Ratio1: 1000, 4 degree overnight in TBST

Secondary Antibody

Secondary DescriptionGoat anti-mouse HRP
Secondary Manufacturer Cat#NBP1-47596
Secondary Concentration1ug/mL
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Lindsy Dickerson
WB Mouse 04/28/2014
View

Summary

ApplicationWestern Blot
Sample TestedMouse
SpeciesMouse
Immunohistochemistry HIF-1 alpha NB100-105
Enlarge
reviewed by:
Ewa Kaniewska
IHC Other 04/14/2014
View

Summary

ApplicationImmunohistochemistry
SpeciesOther
FileView PDF
Immunofluorescence HIF-1 alpha NB100-105
Enlarge
reviewed by:
Ewa Kaniewska
IF Other 04/14/2014
View

Summary

ApplicationImmunofluorescence
SpeciesOther
FileView PDF
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Darlah Lopez
WB Mouse 03/28/2014
View

Summary

ApplicationWestern Blot
SpeciesMouse
Special ApplicationsPrimary 1:1000Secondary 1: 10000
FileView PDF
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Hua Li
WB Human 08/23/2012
View

Summary

ApplicationWestern Blot
Sample TestedWhole cell lysate
SpeciesHuman
LotAB7

Blocking

Blocking Details5% milk in TBS-T buffer, room temperature for one hours

Primary Anitbody

Dilution Ratio1:500 in 5% milk -TBS-T solution, 4C for overnight

Secondary Antibody

Secondary Descriptiongoat anti-mouse IgG, HRP
Secondary Manufacturer Cat#Santa Cruz, Cat# sc-2005
Secondary Concentration1:4000

Details

Detection NotesECL solution for 5min, then exposure to film 3-5min
Immunofluorescence HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
IF Rat 08/22/2012
View

Summary

ApplicationImmunofluorescence
Sample TestedRAT KIDNEY
SpeciesRat
LotF2
CommentsPMID 21270095

Blocking

Blocking Detailsserum blocking for 30 minutes.

Primary Anitbody

Dilution Ratio1:100, overnight incubation,room temperature.

Details

Detection Notes1:1000 Alexa Flour 488, 3:1000 Hoechst 33342.
Fixation Details70% acetone 30% methanol.
Wash Description1xPBS, 5 minutes each,3 times.

Comments

CommentsPMID 21270095
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Camilla Sampaoli
WB Human 08/22/2012
View

Summary

ApplicationWestern Blot
Sample Testedhuman adrenal cancer cells (whole cell lysate)
SpeciesHuman

Blocking

Blocking Details5% non fat dry milk in PBS 1x + 0,1% tween; 1 h at RT

Primary Anitbody

Dilution Ratio1:500; 5% non fat dry milk in PBS 1x + 0,1% tween; o.n. at +4°C

Secondary Antibody

Secondary Descriptiongoat anti-rabbit HRP conjugated
Secondary Manufacturer Cat#Sigma-Aldrich
Secondary Concentration1:4000; 1 h at RT

Details

Detection Notesenhanced chemiluminescence (ECL) assay; exposure: 30 min
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Haofeng Ji
WB Mouse 05/15/2012
View

Summary

ApplicationWestern Blot
Sample TestedCell lysate
SpeciesMouse

Blocking

Blocking Details10% milk

Primary Anitbody

Dilution Ratio1:500

Secondary Antibody

Secondary Descriptionanti-rat IgG
Secondary Concentration1:2000

Details

Detection NotesECL
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Claudia Di Serio
WB Human 05/11/2012
View

Summary

ApplicationWestern Blot
Sample TestedHuman prostate cancer cells
SpeciesHuman
LotAB1
CommentsA good result of Novus' HIF-1 alpha Antibody (NB100-105) was obtained when we used a cell lysate with a plate (10 cm) of LNCaP. The cell line and the exposure time of plate was 20 minutes. We realized an immunoreactive band of about 120 KDa.

Blocking

Blocking Details5% dry milk inTBS 1X/tween (0.1%) buffer

Primary Anitbody

Dilution Ratio1:500, 4 Celcius, O/N

Secondary Antibody

Secondary Descriptionanti-mouse HRP conjugated
Secondary Manufacturer Cat#Pierce Biotechnology
Secondary Concentration1:2000

Details

Detection NotesImmunoreactive bands were visualized by enhanced chemiluminescence (ECL) assay

Comments

CommentsA good result of Novus' HIF-1 alpha Antibody (NB100-105) was obtained when we used a cell lysate with a plate (10 cm) of LNCaP. The cell line and the exposure time of plate was 20 minutes. We realized an immunoreactive band of about 120 KDa.
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Rat 12/14/2011
View

Summary

ApplicationWestern Blot
Sample TestedRat liver
SpeciesRat

Blocking

Blocking DetailsBlocking Buffer: 5% non-fat milk in TBST

Primary Anitbody

Dilution RatioDilution Ratio: 1:500, Incubation Time: overnight, Incubation Temp: 4 degrees Celsius

Secondary Antibody

Secondary DescriptionSecondary Ab: anti-mouse, Secondary Ab Dilution Ratio: 1:10000

Details

Detection NotesDetection Method: ECL
Western Blot HIF-1 alpha NB100-105
Enlarge
reviewed by:
Anonymous
WB Mouse 12/09/2011
View

Summary

ApplicationWestern Blot
Sample TestedMouse auditory cultured cell, Sample Amount: 30ug
SpeciesMouse
CommentsHIF-1 alpha degradation was quick, and our result was still detected by your antibody.

Blocking

Blocking DetailsBlocking Buffer: 1% BSA

Primary Anitbody

Dilution RatioPrimary Ab Dilution Ratio: 1:1000, Primary Ab Incubation Time: 1 hour

Secondary Antibody

Secondary DescriptionSecondary Ab: anti-mouse HRP

Details

Detection NotesDetection Method: Millipore ECL

Comments

CommentsHIF-1 alpha degradation was quick, and our result was still detected by your antibody.
 
reviewed by:
Anonymous
WB Human 11/21/2011
View

Summary

ApplicationWestern Blot
Sample TestedHuman
SpeciesHuman

Blocking

Blocking DetailsBlocking Buffer: 5% non-fat milk in TBST

Primary Anitbody

Dilution RatioPrimary Ab Dilution Ratio: 1:500, Primary Ab Incubation Time: overnight

Secondary Antibody

Secondary DescriptionSecondary Ab: anti-mouse

Details

Detection NotesDetection Method: Wesern Lighting plus ECL
 
reviewed by:
Lu-Hai Wang
ICC Human 11/15/2011
View

Summary

ApplicationImmunocytochemistry
Sample TestedHuman tissues
SpeciesHuman
CommentsIt's specific, but not very intense under current expected conditions.

Blocking

Blocking DetailsBlocking Buffer: BSA

Primary Anitbody

Dilution RatioPrimary Ab Dilution Ratio: 1:2000, Primary Ab Incubation Time: overnight

Secondary Antibody

Secondary DescriptionSecondary Ab: Goat anti-mouse IgG [Alexa Flour 594]

Details

Detection NotesDetection Visualization Method: Fluorescence microscopy

Comments

CommentsIt's specific, but not very intense under current expected conditions.
 
reviewed by:
Anonymous
WB Human 09/30/2011
View

Summary

ApplicationWestern Blot
Sample TestedMCF-7, Sample Amount: 100ug
SpeciesHuman
CommentsHIF-1 alpha has a half-life of approximately 1-5 min in normoxic conditions, so sometimes the failure to detect its protein is due to the postpone during the sample collection. I strongly suggest all the procedures completed entirely on ice and quickest lysis, processing and storage. Even though, this good antibody is still highly recommended. Cells from MCF-7 grown under normoxia or hypoxia were lysed for HIF-1 alpha blotting. For the best signal, we suggest higher amount of protein (80-120ug) and traditional tank (wet) electroblotting protocol. 10v overnight transferring is the longest but most efficient way to transfer big proteins as Hif-1a. 1:500 dilution of this Ab and 1:2000 secondary Ab work well.
Pub Med ID21193031

Blocking

Blocking DetailsBlocking Buffer: 5% Milk in TBST, Blocking Time: 2 hours, Blocking Temp: 20 degrees Celsius

Primary Anitbody

Dilution RatioDilution Ratio: 1:500, Incubation Dilution Buffer: 5% milk TBST, Incubation Time: 16 hours, Incubation Temp: 4 C

Secondary Antibody

Secondary DescriptionSecondary Ab: HRP labeled anti-mouse ab, Secondary Ab Dilution Ratio: 1:2000
Secondary Manufacturer Cat#7076 from cell signaling

Details

Detection NotesDetection Method: HRP, Exposure Time: 1 minute, Specific Bands: 90 kDa

Comments

CommentsHIF-1 alpha has a half-life of approximately 1-5 min in normoxic conditions, so sometimes the failure to detect its protein is due to the postpone during the sample collection. I strongly suggest all the procedures completed entirely on ice and quickest lysis, processing and storage. Even though, this good antibody is still highly recommended. Cells from MCF-7 grown under normoxia or hypoxia were lysed for HIF-1 alpha blotting. For the best signal, we suggest higher amount of protein (80-120ug) and traditional tank (wet) electroblotting protocol. 10v overnight transferring is the longest but most efficient way to transfer big proteins as Hif-1a. 1:500 dilution of this Ab and 1:2000 secondary Ab work well.
 
reviewed by:
Anonymous
WB Rat 09/01/2010
View

Summary

ApplicationWestern Blot
Sample Testedendothelial cell nuclear extract, Sample Amount: 40ug
SpeciesRat
CommentsGood results using this Ab for Western blots and ChIP analysis.

Blocking

Blocking DetailsBlocking Buffer: PBS + 5% NFDM + 0.05% Tween 20, Blocking Time: 1 hour, Blocking Temp: room temperature

Primary Anitbody

Dilution RatioDilution Ratio: 1:250, Incubation Dilution Buffer: PBS + 5% NFDM + 0.05% Tween 20, Incubation Time/Temp: overnight at 4C

Secondary Antibody

Secondary DescriptionSecondary Ab: GaM=HRP, Secondary Ab Dilution Ratio: 1:7500

Details

Detection NotesMethod: Chemiluminescence, Exposure Time: 15 min, Positive Control: nuclear extract from same cells, Specific Bands: 60, 98 kDa

Comments

CommentsGood results using this Ab for Western blots and ChIP analysis.
 
reviewed by:
William Lin
WB Mouse 05/17/2010
View

Summary

ApplicationWestern Blot
Sample Testedmouse cortical neurons, Sample Amount: 40ug
SpeciesMouse
LotAA1
CommentsI tested this Hif1a antibody on mouse primary cortical neuronal culture treated with 100uM DFO for over 4 hours. I collected the total lysate using RIPA buffer with 0.1% SDS, sonicated the lysates, and heated up to 90ºC for 5 minutes in SDS-containing sample buffer before loading on an SDS-PAGE gel. I used 1:1000 dilution for the Hif1a antibody (overnight), and 1:20000 anti-mouse secondary antibody for 1 hour. With a few background bands, this antibody works well to detect the upregulation of Hif1a under hypoxic conditions.

Blocking

Blocking DetailsBlocking Buffer: 5% milk in TBST, Blocking Time: 2 hours, 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-mouse IgG-HRP, Secondary Ab Dilution Ratio: 1:20000
Secondary Manufacturer Cat#Promega W402B

Details

Detection NotesDetection Method: ECL, Exposure Time: 2 min, Neg.Control: untreated neurons, Specific Bands: 120 kDa, Non-Specific Bands: 67 kDa

Comments

CommentsI tested this Hif1a antibody on mouse primary cortical neuronal culture treated with 100uM DFO for over 4 hours. I collected the total lysate using RIPA buffer with 0.1% SDS, sonicated the lysates, and heated up to 90ºC for 5 minutes in SDS-containing sample buffer before loading on an SDS-PAGE gel. I used 1:1000 dilution for the Hif1a antibody (overnight), and 1:20000 anti-mouse secondary antibody for 1 hour. With a few background bands, this antibody works well to detect the upregulation of Hif1a under hypoxic conditions.
 
reviewed by:
Anonymous
ICC Other 02/10/2010
View

Summary

ApplicationImmunocytochemistry
Sample TestedHUVEC & Candida albicans
SpeciesOther
LotZ5
CommentsWorks fine with HUVEC but not when Candida albicans is present. The Candida hyphae bind the Ab w/ much greater affinity than the EC and act as a sink leaving the EC completely unstained.

Blocking

Blocking DetailsBlocking Buffer: PBST +1% BSA, Blocking Time: 30 minutes, Blocking Temp: 25 degrees Celsius

Primary Anitbody

Dilution RatioIncubation Dilution Buffer: PBST + 1% BSA, Dilution Ratio: 1/100, Incubation Time: 18 hours, Incubation Temp: 4 degrees Celsius

Secondary Antibody

Secondary DescriptionSecondary Ab: anti-mouse IgG, Secondary Ab Dilution: 1/1000
Secondary Manufacturer Cat#Invitrogen A11004

Comments

CommentsWorks fine with HUVEC but not when Candida albicans is present. The Candida hyphae bind the Ab w/ much greater affinity than the EC and act as a sink leaving the EC completely unstained.

Product General Protocols

Video Protocols

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

FAQs for HIF-1 alpha Antibody (NB100-105). (Showing 1 - 10 of 30 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 30 FAQs.