HIF-2 alpha/EPAS1 Antibody

Images

 
Genetic Strategies: Western Blot: HIF-2 alpha/EPAS1 Antibody [NB100-122] - Western blot of 786-O cells without or with VHL overexpression. Image from verified customer review.
Independent Antibodies: Western Blot: HIF-2 alpha/EPAS1 Antibody [NB100-122] - Analysis of HIF-2 alpha in MDA-MB-231 cell lysate (overexpression and endogenous samples) using anti-HIF-2 alpha antibody. The data ...read more
Immunocytochemistry/ Immunofluorescence: HIF-2 alpha/EPAS1 Antibody [NB100-122] - HIF-2 alpha antibody was tested in RCC4 cells at a 1:200 dilution against Dylight 488 (Green). Alpha tubulin and nuclei were ...read more
Biological Strategies: Western Blot: HIF-2 alpha/EPAS1 Antibody [NB100-122] - Analysis of HIF-2 alpha in human Retinal and Choroidal primary endothelia lysates using anti-HIF-2 alpha antibody. Image from verified ...read more
Biological Strategies: Western Blot: HIF-2 alpha/EPAS1 Antibody [NB100-122] - Analysis using the HRP conjugate of NB100-122. Detection of normoxic and hypoxic nuclear rat cell lysates.
Biological Strategies: Western Blot: HIF-2 alpha/EPAS1 Antibody [NB100-122] - Analysis of HIF-2 alpha on Lane 1, Cobalt chloride treated COS7 nuclear extracts and Lane 2, Untreated COS7 nuclear extracts using ...read more
Immunohistochemistry-Paraffin: HIF-2 alpha/EPAS1 Antibody [NB100-122] - Hif-2 alpha immunoreactivity in human cardiac myocytes stained with NB100-122.
Biological Strategies: Western Blot: HIF-2 alpha/EPAS1 Antibody [NB100-122] - Analysis on normoxic and hypoxic nuclear rat cell lysates.
Western Blot: HIF-2 alpha/EPAS1 Antibody [NB100-122] - Western Blot on Human Glioma cells, whole cell lysates from a customer review.
Simple Western: HIF-2 alpha/EPAS1 Antibody [NB100-122] - Simple Western lane view shows a specific band for HIF-2 alpha in 0.5 mg/ml of Hypoxic HeLa lysate. This experiment was performed under reducing conditions ...read more

Product Details

Summary
Reactivity Hu, Mu, Rt, Fi, Ha, Pm, ShSpecies Glossary
Applications WB, Simple Western, ChIP, ELISA, Flow, GS, ICC/IF, IHC, IHC-Fr, IHC-P, IP, KO
Clonality
Polyclonal
Host
Rabbit
Conjugate
Unconjugated
Concentration
1.0 mg/ml

HIF-2 alpha/EPAS1 Antibody Summary

Immunogen
A peptide derived from the C-terminus of mouse/human HIF-2 alpha protein.
Specificity
Specific for HIF-2 alpha/EPAS. Does not cross-react with HIF-1 alpha.
Isotype
IgG
Clonality
Polyclonal
Host
Rabbit
Gene
EPAS1
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 1.0 ug/ml - 2.0 ug/ml
  • Simple Western 1:50
  • Chromatin Immunoprecipitation 1:10 - 1:500
  • ELISA 1:100 - 1:2000
  • Flow Cytometry
  • Gel Super Shift Assays
  • Immunocytochemistry/Immunofluorescence 1:100
  • Immunohistochemistry 1:100
  • Immunohistochemistry-Frozen
  • Immunohistochemistry-Paraffin 1:100
  • Immunoprecipitation 5ug/1mg lysate
  • Knockout Validated
Application Notes
This HIF-2 alpha antibody is useful for Western blot, ICC/IHC, IP, ChIP, ELISA, Gel Shift Super Assay (PMID: 15184875), Use in Immunoblotting reported in scientific literature (PMID 28115701). In WB, this antibody recognizes a band at 118kDa representing HIF-2 alpha. 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).
Theoretical MW
118 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.
Positive Control
PC-12 Lysate (NB810-55229)
COS-7 Lysate (NB800-PC26)
Rectum Lysate (NB820-59250)
HIF-2 alpha/EPAS1 Lysate (NBP2-64985)
Reviewed Applications
Read 26 Reviews rated 4.4
using
NB100-122 in the following applications:

Publications
Read Publications using
NB100-122 in the following applications:

  • 39 publications
  • 1 publication
  • 5 publications
  • 3 publications
  • 1 publication
  • GS
    3 publications
  • IB
    20 publications
  • 18 publications
  • IHC
    21 publications
  • 4 publications
  • 29 publications
  • IP
    17 publications
  • WB
    341 publications

Reactivity Notes

Reactivity with sheep reported in the scientific literature (PMID: 17307811). Hamster reactivity reported in scientific literature (PMID: 24997364 and 24997360)

Packaging, Storage & Formulations

Storage
Store at -20 °C.
Buffer
PBS
Preservative
0.05% Sodium Azide
Concentration
1.0 mg/ml
Purity
Immunogen affinity purified

Alternate Names for HIF-2 alpha/EPAS1 Antibody

  • anti hif 2 alpha
  • anti-HIF-2 alpha
  • Basic-helix-loop-helix-PAS protein MOP2
  • BHLHE73
  • bHLHe73ECYT4
  • Class E basic helix-loop-helix protein 73
  • endothelial PAS domain protein 1
  • endothelial PAS domain-containing protein 1
  • EPAS1
  • EPAS-1
  • HIF 2A
  • HIF-1alpha-like factor
  • HIF2 alpha
  • HIF-2 alpha
  • hif2a angiogenesis
  • HIF2A
  • HIF2AHIF-2-alpha
  • HLF
  • HLFHIF-1-alpha-like factor
  • hypoxia-inducible factor 2 alpha
  • Hypoxia-inducible factor 2-alpha
  • Member of PAS protein 2
  • MOP2
  • MOP2HIF2-alpha
  • PASD2PAS domain-containing protein 2

Background

Hypoxia-inducible factors (HIFs) generate heterodimers composed of alpha-subunits (HIF-1 alpha, HIF-2 alpha and HIF-3 alpha) and beta subunits (Arnt1, Arnt2 and Arnt3). Of these isoforms, HIF-1 alpha and HIF-2 alpha play critical roles in the cellular response to hypoxic conditions. After heterodimerization with beta subunits binding to DNA sequence 5'-[AG]CGTG-3' within HRE (hypoxia response element) of target gene promoters, HIF-2 alpha regulates the gene expression in response to oxygen homeostasis alterations. HIF-2 alpha regulates VEGF expression involved in the development of new blood vessels as well as the tubular system of the lung. HIF-2 alpha plays a role in the formation of the endothelium that gives rise to blood brain barrier. HIF-2 alpha is a strong activator of expression of Tie-2 tyrosine kinase, which is a major player in angiogenesis. HIF-2 alpha activation requires recruitment of transcriptional coactivators such as CREBPB, EP300, and its interaction with redox regulatory protein APEX mediates activation of CTAD. HIF-2 alpha and HIF-1 alpha expression increases in certain malignancies; wherein, HIF-1 alpha plays both pro- as well as anti-proliferative activities. HIF-2 alpha is suggested to exert anti-proliferative activity only.

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-2 alpha/EPAS1 Antibody (NB100-122)(518)

We have publications tested in 7 confirmed species: Human, Mouse, Rat, Hamster, Mouse , Rat , Sheep.

We have publications tested in 13 applications: ChIP, Co-IP, ELISA, FLOW, Flow, GS, IB, ICC/IF, IHC, IHC-Fr, IHC-P, IP, WB.


Filter By Application
ChIP
(39)
Co-IP
(1)
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(5)
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(3)
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(1)
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(3)
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(20)
ICC/IF
(18)
IHC
(21)
IHC-Fr
(4)
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(29)
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(17)
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(341)
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Human
(275)
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(121)
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(25)
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(2)
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(1)
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(1)
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Showing Publications 1 - 10 of 518. Show All 518 Publications.
Publications using NB100-122 Applications Species
Robinson CM, Lefebvre F, Poon BP, Bousard A. Consequences of VHL Loss on Global DNA Methylome. Sci Rep. 2018 Feb 20 [PMID: 29463811] (GS, Human) GS Human
Senavirathna LK, Huang C, Yang X, Munteanu MC. Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling. Sci Rep. 2018 Feb 09 [PMID: 29426911] (WB, Human) WB Human
Mira E, Carmona-Rodriguez L, Perez-Villamil B, Casas J. SOD3 improves the tumor response to chemotherapy by stabilizing endothelial HIF-2a. Nat Commun. 2018 Feb 08 [PMID: 29422508] (WB, Human) WB Human
Frump AL, Selej M, Wood JA, Albrecht M. Hypoxia Up-regulates Estrogen Receptor b in Pulmonary Artery Endothelial Cells in a HIF-1a Dependent Manner. Am. J. Respir. Cell Mol. Biol. 2018 Feb 02 [PMID: 29394091] (Rat) Rat
Liu S, Tetzlaff MT, Wang T et al. Hypoxia-activated prodrug enhances therapeutic effect of sunitinib in melanoma Oncotarget 2017 Dec 29 [PMID: 29383148] (WB, Mouse) WB Mouse
Samanta D, Park Y, Ni X et al. Chemotherapy induces enrichment of CD47+/CD73+/PDL1+ immune evasive triple-negative breast cancer cells Proc. Natl. Acad. Sci. U.S.A. 2018 Jan 24 [PMID: 29367423] (ChIP, Human) ChIP Human
Bogurcu N, Seidel S, Garvalov BK, Acker T. Analysis of Hypoxia and the Hypoxic Response in Tumor Xenografts Methods Mol. Biol. 2018 Jan 14 [PMID: 29330809] (WB) WB
Brooks DL, Seagroves TN. Chromatin Immunoprecipitation of HIF-a in Breast Tumor Cells Using Wild Type and Loss of Function Models Methods Mol. Biol. 2018 Jan 14 [PMID: 29330791] (IP) IP
Ho JJD, Balukoff NC, Cervantes G, Malcolm PD. Oxygen-Sensitive Remodeling of Central Carbon Metabolism by Archaic eIF5B. Cell Rep. 2018 Jan 02 [PMID: 29298419] (WB) WB
Backer V, Cheung FY, Siveke JT et al. Knockdown of myeloid cell hypoxia-inducible factor-1a ameliorates the acute pathology in DSS-induced colitis. PLoS ONE 2017 Dec 20 [PMID: 29261815] (IHC-P, Mouse) IHC-P Mouse
Show All 518 Publications.

Reviews for HIF-2 alpha/EPAS1 Antibody (NB100-122) (26) 4.426

Average Rating: 4.4
(Based on 26 reviews)
We have 26 reviews tested in 4 species: Human, Mouse, Rat, Other.

Reviews using NB100-122:
Filter by Applications
WB
(22)
ChIP
(2)
IHC-P
(1)
IF
(1)
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Filter by Species
Human
(17)
Mouse
(5)
Rat
(1)
Other
(2)
All Species
Images Ratings Applications Species Date Details
 
reviewed by:
Shanhai Xie
WB Mouse 01/12/2017
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Summary

ApplicationWestern Blot
Sample Testedtransfected U2OS cell lysate
SpeciesMouse
LotBV-2
 
reviewed by:
Anonymous
WB Human 01/12/2017
View

Summary

ApplicationWestern Blot
Sample Testedcultured cells
SpeciesHuman
LotBV-2
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Anonymous
WB Mouse 10/18/2016
View

Summary

ApplicationWestern Blot
Sample TestedMouse CD4+ T Cells
SpeciesMouse
LotBP-1

Comments

CommentsDilution (1:1000)
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Helder Andre
WB Human 07/11/2016
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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+
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Anonymous
WB Human 03/31/2016
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Summary

ApplicationWestern Blot
Sample TestedHCT116 cell line lysate
SpeciesHuman
LotBP-2

Blocking

Blocking Details5% skim milk in TBS

Primary Anitbody

Dilution Ratio1000 times, overnight incubation at 4 degrees C in 0.1% Tween-20 TBS 5% milk

Secondary Antibody

Secondary DescriptionGE Healthcare Anti-rabbit IgG HRP
Secondary Manufacturer Cat#NA934V
Secondary Concentration5,000 dilution

Details

Detection NotesDetection using autoradiography film, exposure 1 minutes, 24 hours hypoxia as positive controls.
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Anonymous
WB Human 11/10/2015
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Summary

ApplicationWestern Blot
Sample Testedcytoplasmic and nuclear protein fractions of U266 cells
SpeciesHuman

Blocking

Blocking Details5% milk in PBST, 30 minutes at Room Temperature

Primary Anitbody

Dilution Ratio1 ug/ml, Overnight, 4 C, PBST with 5%milk

Secondary Antibody

Secondary DescriptionGoat anti-rabbit igG HRP
Secondary Manufacturer Cat#SC-2005
Secondary Concentration1:2000

Details

Detection NotesChemiluminescence, G:Box Chemi XLT4, 12 sec, no positive and negative controls

Comments

CommentsThis antibody showed clean bands by wet transfer method.
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Anonymous
WB Human 08/12/2015
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Summary

ApplicationWestern Blot
Sample TestedPrimary Epithelial Cells
SpeciesHuman

Blocking

Blocking Details5% Milk in 0.1% TBST for 1 hour at room temperature

Primary Anitbody

Dilution Ratio1:500, Overnight at 4 celcius in 5% milk in 0.1% TBST

Secondary Antibody

Secondary DescriptionGoat anti-rabbit HRP
Secondary Manufacturer Cat#KPL 0741516
Secondary Concentration1:5000

Details

Detection NotesChemiluminescence, BioRad Universal Hood II, 10 minute exposure
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Anonymous
WB Human 06/01/2015
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Summary

ApplicationWestern Blot
Sample TestedMDA-MB-231 Cell Lysate
SpeciesHuman
LotBQ1

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 HIF1a-overexpression as shown in the picture)
 
reviewed by:
Anonymous
WB Human 03/27/2015
View

Summary

ApplicationWestern Blot
Sample Tested786O cell lysates
SpeciesHuman

Blocking

Blocking Details5% milk in TBST

Primary Anitbody

Dilution Ratio1:1000

Secondary Antibody

Secondary DescriptionGoat anti-rabbit
Secondary Concentration1:5000

Details

Detection Notesusing the regular pico from pierece, detection by Bio-rad imager, 2 min exposure time, 786O vector cells is the positive ctrl.
 
reviewed by:
Anonymous
ChIP Human 12/12/2014
View

Summary

ApplicationChromatin Immunoprecipitation
Sample TestedSee PMID:24248342
SpeciesHuman

Details

Detection NotesSee PMID:24248342

Comments

CommentsPublished in PMID:24248342
 
reviewed by:
Anonymous
WB Human 12/12/2014
View

Summary

ApplicationWestern Blot
Sample TestedSee PMID:24308012
SpeciesHuman

Blocking

Blocking DetailsSee PMID:24308012

Details

Detection NotesAntibody works well but long exposure times are needed.

Comments

CommentsPublished in PMID:24308012
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Xiuquan Luo
WB Human 09/12/2014
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Summary

ApplicationWestern Blot
Sample TestedHEK293 cell lysate
SpeciesHuman

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
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Pramod Mallikarjuna
WB Human 08/30/2014
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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-Rat IgG
Secondary Manufacturer Cat#926-32219
Secondary Concentration1:10000

Details

Detection NotesOdyssey® CLx Infrared Imaging System
 
reviewed by:
Anonymous
WB Human 06/30/2014
View

Summary

ApplicationWestern Blot
Sample TestedWhole cell lysate
SpeciesHuman
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Anonymous
WB Human 04/30/2014
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Summary

ApplicationWestern Blot
Sample Testedwhole cell lysate U87MG (human glioblastoma cells)
SpeciesHuman

Blocking

Blocking Details3% milk/TBST, 1h, room temperature

Primary Anitbody

Dilution Ratio1:1000 in 3% milk/TBST, 2h, room temperature

Secondary Antibody

Secondary Descriptiongoat anti-rabbit IgG, HRP
Secondary Manufacturer Cat#Dako
Secondary Concentration1:2000

Details

Detection NotesECL, X-ray film, 1h exposure
Immunohistochemistry-Paraffin HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Saori Yoshii
IHC-P Mouse 04/11/2014
View

Summary

ApplicationImmunohistochemistry-Paraffin
Sample TestedMouse Intestine
SpeciesMouse
LotBP-1
CommentsHIF-2a can be detected in the nucleus. Background in the cytosol is also detected (HIF-2a real band only appeared in the nuclear fraction, checked by WB).

Blocking

Blocking DetailsInvitrogen HistoMouse kit

Primary Anitbody

Dilution Ratiox300 in 1% BSA

Secondary Antibody

Secondary DescriptionInvitrogen HistoMouse kit

Details

Detection NotesDAB
Fixation Details+antigen retrieval

Comments

CommentsHIF-2a can be detected in the nucleus. Background in the cytosol is also detected (HIF-2a real band only appeared in the nuclear fraction, checked by WB).
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Anonymous
WB Human 04/03/2014
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Summary

ApplicationWestern Blot
SpeciesHuman
LotBP-1
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Prabir Chakraborty
WB Human 01/24/2014
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Summary

ApplicationWestern Blot
Sample TestedUNTREATED OVARIAN SURFACE EPITHELIUM CELLS (OSE) AND A2780 CELLS LYSATES 25ug
SpeciesHuman
LotBB1

Blocking

Blocking Details4% BSA IN TBST, 1H ROOM TEMPERATURE

Primary Anitbody

Dilution Ratio1:750 IN 4% BSA IN TBST FOR O/N AT 4*C

Secondary Antibody

Secondary DescriptionGoat anti-rabbit IgG-HRP
Secondary Manufacturer Cat#sc-2301
Secondary Concentration1:10,000

Details

Detection NotesECL
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Anonymous
WB Human 11/20/2013
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Summary

ApplicationWestern Blot
Sample TestedHuman Glioma cells, whole cell lysates
SpeciesHuman
LotBN-1

Blocking

Blocking DetailsMilk 5% in Tween TBS, 30 min, 25°C

Primary Anitbody

Dilution Ratio1/500, 16 hours at 4°C in Milk 1% in TBS Tween 0.1%

Secondary Antibody

Secondary DescriptionGoat Anti-rabbit IgG (H+L)
Secondary Manufacturer Cat#Bio-Rad
Secondary Concentration1/10000

Details

Detection NotesECL
Immunofluorescence HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Anonymous
IF Mouse 04/15/2013
View

Summary

ApplicationImmunofluorescence
SpeciesMouse
Pub Med ID22753893
FileView PDF
Chromatin Immunoprecipitation HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Anonymous
ChIP Rat 04/02/2013
View

Summary

ApplicationChromatin Immunoprecipitation
SpeciesRat
Special ApplicationsRat duodenal enterocyte
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Anonymous
WB 09/19/2012
View

Summary

ApplicationWestern Blot
LotBK-2
Pub Med ID21288809
FileView PDF
 
reviewed by:
Anonymous
WB Mouse 12/08/2011
View

Summary

ApplicationWestern Blot
Sample TestedMouse
SpeciesMouse

Blocking

Blocking Details1Blocking Buffer: 0% Milk

Primary Anitbody

Dilution RatioDilution Ratio: 1:1000, Incubation Time: overnight, Incubation Temp: 4 degrees Celsius

Secondary Antibody

Secondary DescriptionSecondary Ab Dilution Ratio: 1:5000

Details

Detection NotesDetection Method: ECL
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Olga Razorenova
WB Human 06/15/2011
View

Summary

ApplicationWestern Blot
Sample TestedACHN, SN12C, Caki1 whole cell lysates, Sample Amount: 30ug
SpeciesHuman
LotBC3
CommentsAntibody at 1:500 dilution detects HIF2 alpha at the appropriate molecular weight (around 94kDa) in cells exposed to hypoxia (0.5% O2, 16h). No need to do nuclear extracts, it works for total cell protein extracts, also the amount of protein for lane should be 30-80ug. Usually 30 ug is enough.





Good luck!!!
Pub Med ID21233420

Blocking

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

Primary Anitbody

Dilution RatioDilution Ratio: 1:500, Incubation Dilution Buffer: 5% Milk in PBST, Incubation Time: overnight, Incubation Temp: 4 C

Secondary Antibody

Secondary DescriptionSecondary Ab: anti-Rabbit-HRP from Pierce, Secondary Ab Dilution Ratio: 1:5000
Secondary Manufacturer Cat#31460

Details

Detection NotesMethod: ECL, Exposure Time: 3 min, Pos.Ctrl: Hypoxia treatment, Neg.Ctrl: Normoxia (21% O2) treatment, Specific Bands: 94 kDa

Comments

CommentsAntibody at 1:500 dilution detects HIF2 alpha at the appropriate molecular weight (around 94kDa) in cells exposed to hypoxia (0.5% O2, 16h). No need to do nuclear extracts, it works for total cell protein extracts, also the amount of protein for lane should be 30-80ug. Usually 30 ug is enough. Good luck!!!
Western Blot HIF-2 alpha/EPAS1 NB100-122
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reviewed by:
Anonymous
WB Other 03/11/2010
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Summary

ApplicationWestern Blot
Sample TestedColon Cancer Cell llines, Sample Amount: 50ug
SpeciesOther
LotAT1
CommentsThe image was of RKO cells. Using HCT116 cells we saw multiple bands, but the band at 118 was deinitely stronger. We image out westerns using a Tyhpoon and ECL Plus. We find we can use more dilute antibodies than when we were using regular ECL and film.

Blocking

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

Primary Anitbody

Dilution RatioDilution Ratio: 1/2000, Incubation Dilution Buffer: 5% BSA in TNS with Tween, Incubation Time: overnight, Incubation Temp: 4 C

Secondary Antibody

Secondary DescriptionSecondary Ab: anti-Rabbit, Secondary Ab Dilution Ratio: 1/10000

Details

Detection NotesDetection Method: ECL Plus, Specific Bands: 118 kDa

Comments

CommentsThe image was of RKO cells. Using HCT116 cells we saw multiple bands, but the band at 118 was deinitely stronger. We image out westerns using a Tyhpoon and ECL Plus. We find we can use more dilute antibodies than when we were using regular ECL and film.
 
reviewed by:
Anonymous
WB Other 02/09/2009
View

Summary

ApplicationWestern Blot
Sample Testedhead and neck squamous cell carcinoma cells, Sample Amount: 35 ug
SpeciesOther

Blocking

Blocking DetailsBlocking Buffer: 5% nonfat dry milk in TBS-T, Blocking Time: 1 hour, Blocking Temp: 25 degrees Celsius

Primary Anitbody

Dilution RatioDilution Ratio: 1/500, Incubation Dilution Buffer: 5% nonfat dry milk in TBS-T, Incubation Time: overnight, Incubation Temp: 4 C

Secondary Antibody

Secondary DescriptionSecondary Ab: rabbit polyclonal HRP, Secondary Ab Dilution Ratio: 1/10000

Details

Detection NotesDetection Method: ECL, Exposure Time: varies, Pos.Ctrl: hypoxia-treated sample, Neg.Ctrl: normoxia, Specific Bands: ~120 kDa

Product General Protocols

Video Protocols

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

FAQs for HIF-2 alpha/EPAS1 Antibody (NB100-122). (Showing 1 - 10 of 10 FAQs).

  1. What is the synthetic peptide of NB100-122, corresponding to which amino acids? Can this antibody cross-react with chicken or not?
    • Unfortunately we cannot provide any further information on this epitope as it is considered proprietary. The immunogen is a peptide derived from the C-terminus of mouse/human HIF-2 alpha protein. I have carried out a blast for the % homology between human HIF-2 alpha and Chicken HIF-2 alpha. The % homology is 75% and our lab only recommends use when the homology is 85% or above.
  2. What do you recommend for blocking for a Western blot with this antibody?
    • We recommend to block membranes for 1.5 hours with 1X western wash buffer containing 5% non-fat dry milk (NFDM). For more information, a product specific WB protocol can be found on the datasheet if you click on the "Protocols & FAQs" tab.
  3. We are using HIF2 Antibody from Novus to detect HIF2 in hypoxic HUVEC cells. What washing buffer do you use for Western Blot with HIF2? Are you using PBS or TBS, 0.3 or 0.1% Tween to solute the 5% milk (NFDM)? Do you solute the antibody 1:500 or 1:1000?
    • Please see our specific protocol for Hif2 here. We recommend 0.5 to 1.0% Tween as a washing buffer and we recommend a dilution of 1:200 to 1:1000. This is depending on your sample and you will have to optimize this accordingly.
  4. I am working on a WB using the antibody against mouse HIF1a and HIF2a (catalog numbers NB100-105 and NB100-122). I tried several anti-mouse and anti-rabbit HRP secondary antibodies (from another company) and found they gave different patterns. Please provide the secondary antibody info which was used in the WB showed on the datasheet.
    • We use catalog numbers NB7570 and NB7183 as our secondary antibodies for testing these antibodies in WB.
  5. Is this antibody useful for EMSA-supershift?
    • Unfortunately we have not tested if this antibody can be used in EMSA-supershift. If you would be interested in testing this novel application, please take a look at our Innovator's Reward program.
  6. Will the hif protein be degraded when preparing the nuclear extract?
    • Working with Hif-2 can be tricky at times and the possibility of degradation is a very real one. Please see this link for a specific protocol for using NB100-122. The nuclear extract protocol can be found using this reference: Wang and Semenza. Purification and Characterization of Hypoxia-Inducible Factor 1. Journal of Biological Chemistry. 270(3): 1230-1237, 1995. I would also recommend that you use a positive control; a nuclear extract of hypoxia induced cell lines (293, Hep3B, COS7, Hepa) are all excellent choices. You can purchase the hypoxic COS7 lysate from us to make it more convenient - the catalog number is NB800-PC26. Please also see our Hypoxia support page for suggestions.
  7. We've purchased NB100-122 for Western blot and IHC. I'd like to know what would be good positive control for this antibody in case of IHC.
    • For HIF2 alpha positive control tissues, we recommend the following: Kidney, Ling, Brain, Heart. For western blot, we recommend using a positive and negative control such as normoxic/hypoxic or treated/untreated with CoCl2. We sell a CoCl2 treated/untreated Cos7 lysate: View Cos7 lysate datasheet.
  8. What is the sequence for the peptide used to purify this antibody?
    • The exact sequence of the immunogen is proprietary, but I can tell you the range is within 625-695.
  9. Could you tell us if these antibodies need to use cell lysate treated with special extraction for WB? Do you have the recommended preparation method of cell lysate?
    • Usually HIF1 alpha and HIF2 alpha are hard to detect endogenously and require some inducement. The most common is hypoxic treatment of the cells. This can be also be chemically induced by CoCl2 treatment as well. Our recommended protocols and lysate preparations can be found in our white paper here: http://www.novusbio.com/whitepapers/cellular-response-hypoxia.html.
  10. For paraffin sections, do you recommend the antigen retrieval?
    • Unless specially noted in the protocol section we always recommend using citrate buffer pH 6 for antigen retrieval. 

Positive Control Lysate(s)

Secondary Antibodies

 

Isotype Controls

Other Available Formats

Alexa Fluor 405 Labeled NB100-122AF405
Alexa Fluor 488 Labeled NB100-122AF488
Alexa Fluor 647 Labeled NB100-122AF647
Alexa Fluor 700 Labeled NB100-122AF700
Biotin Labeled NB100-122B
DyLight 405 Labeled NB100-122V
DyLight 488 Labeled NB100-122G
DyLight 550 Labeled NB100-122R
DyLight 650 Labeled NB100-122C
FITC Labeled NB100-122F
HRP Labeled NB100-122H
PE Labeled NB100-122PE

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Blogs on HIF-2 alpha/EPAS1.

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.

HIF-2 alpha, Tumor Suppression and Cell Survival
HIF-2 alpha is one subunit within the HIF-2 nuclear protein that regulates cellular responses to hypoxia (low oxygen tension conditions). Hydroxylation post-translational modifications on particular HIF residues target them for degradation. Luo, et al...  Read full blog post.

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Shanhai Xie
01/12/2017
Application: WB
Species: Mouse

 
Anonymous
01/12/2017
Application: WB
Species: Human

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10/18/2016
Application: WB
Species: Mouse

Bioinformatics

Gene Symbol EPAS1
Entrez