Anti-HIF-2-alpha抗体- ChIP Grade (ab199)

概述

  • 产品名称
    Anti-HIF-2-alpha抗体- ChIP Grade
    参阅全部 HIF-2-alpha 一抗
  • 描述
    兔多克隆抗体to HIF-2-alpha - ChIP Grade
  • 宿主
    Rabbit
  • 特异性
    Specific for HIF-2-alpha / EPAS 1. Does not cross react with HIF-1-alpha.
  • 经测试应用
    适用于: Flow Cyt, IHC-Fr, ChIP, IP, ICC/IF, IHC-P, WBmore details
  • 种属反应性
    与反应: Mouse, Rat, Human
  • 免疫原

    Synthetic peptide corresponding to Mouse HIF-2-alpha aa 632-646.
    Sequence:

    GRSNTQWPPDPPLHF

  • 阳性对照
    • CoCl2-treated Cos7 nuclear extract or hypoxic/normoxic PC12 nuclear extracts. Under normoxic conditions HIF-2 alpha has a short half-life. It is largely undetectable in cells or tissues grown under normoxic conditions. It is stabilized only at O2 concentrations below 5% and upon stabilization under hypoxic conditions HIF-2 translocate to the nucleus. Therefore we recommend western blots using nuclear extracts and running Hypoxia treated samples as positive control (ab180880).

性能

应用

Our Abpromise guarantee covers the use of ab199 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用 Ab评论 说明
Flow Cyt Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
ChIP Use at an assay dependent concentration. PubMed: 19897487
IP Use at 5-1 µg/mg of lysate.
ICC/IF Use a concentration of 1 µg/ml.
IHC-P 1/100.
EMSA Use at an assay dependent concentration.
WB 1/200 - 1/1000. Detects a band of approximately 118 kDa (predicted molecular weight: 100 kDa). The following should be taken into consideration: sufficient hypoxia induction is necessary; use nuclear extracts for cleaner results; use positive/negative controls to see which band is up-regulated; HIF2a degrades rapidly (fast sample preparation, protease inhibitors!).

靶标

  • 功能
    Transcription factor involved in the induction of oxygen regulated genes. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Regulates the vascular endothelial growth factor (VEGF) expression and seems to be implicated in the development of blood vessels and the tubular system of lung. May also play a role in the formation of the endothelium that gives rise to the blood brain barrier. Potent activator of the Tie-2 tyrosine kinase expression. Activation seems to require recruitment of transcriptional coactivators such as CREBPB and probably EP300. Interaction with redox regulatory protein APEX seems to activate CTAD.
  • 组织特异性
    Expressed in most tissues, with highest levels in placenta, lung and heart. Selectively expressed in endothelial cells.
  • 疾病相关
    Defects in EPAS1 are the cause of erythrocytosis familial type 4 (ECYT4) [MIM:611783]. ECYT4 is an autosomal dominant disorder characterized by increased serum red blood cell mass, elevated hemoglobin concentration and hematocrit, and normal platelet and leukocyte counts.
  • 序列相似性
    Contains 1 basic helix-loop-helix (bHLH) domain.
    Contains 1 PAC (PAS-associated C-terminal) domain.
    Contains 2 PAS (PER-ARNT-SIM) domains.
  • 翻译后修饰
    In normoxia, is probably hydroxylated on Pro-405 and Pro-531 by EGLN1/PHD1, EGLN2/PHD2 and/or EGLN3/PHD3. The hydroxylated prolines promote interaction with VHL, initiating rapid ubiquitination and subsequent proteasomal degradation. Under hypoxia, proline hydroxylation is impaired and ubiquitination is attenuated, resulting in stabilization.
    In normoxia, is hydroxylated on Asn-847 by HIF1AN thus probably abrogating interaction with CREBBP and EP300 and preventing transcriptional activation.
    Phosphorylated on multiple sites in the CTAD.
    The iron and 2-oxoglutarate dependent 3-hydroxylation of asparagine is (S) stereospecific within HIF CTAD domains.
  • 细胞定位
    Nucleus.
  • Information by UniProt
  • 数据库链接
  • 别名
    • Basic helix loop helix PAS protein MOP2 antibody
    • Basic-helix-loop-helix-PAS protein MOP2 antibody
    • bHLHe73 antibody
    • Class E basic helix-loop-helix protein 73 antibody
    • ECYT4 antibody
    • Endothelial PAS domain containing protein 1 antibody
    • Endothelial pas domain protein 1 antibody
    • Endothelial PAS domain-containing protein 1 antibody
    • EPAS 1 antibody
    • EPAS-1 antibody
    • EPAS1 antibody
    • EPAS1_HUMAN antibody
    • HIF 1 alpha like factor antibody
    • HIF 2 alpha antibody
    • HIF-1-alpha-like factor antibody
    • HIF-2-alpha antibody
    • HIF2-alpha antibody
    • HIF2A antibody
    • HLF antibody
    • Hypoxia inducible factor 2 alpha antibody
    • Hypoxia inducible factor 2 alpha subunit antibody
    • Hypoxia-inducible factor 2-alpha antibody
    • Member of PAS protein 2 antibody
    • Member of pas superfamily 2 antibody
    • MOP 2 antibody
    • MOP2 antibody
    • PAS domain-containing protein 2 antibody
    • PASD2 antibody
    see all

图片

  • Analysis on normoxic and hypoxic nuclear rat cell lysates.

  • ab199 staining human adrenal tissue sections by IHC-P.  Sections were PFA fixed and subjected to heat mediated antigen retrieval in citrate buffer (pH 6) prior to blocking in 10% serum for 10 minutes at 25°C.  The primary anitbody was diluted 1/500 and incubated with the sample for 1 hour at 25°C.  A biotinylated goat anti-rabbit antibody was used as the secondary.

    See Abreview

  • HIF-2α levels in HIF-2α−/− and WT mouse kidneys.

    HIF-2α−/− mice or their wild-type littermates were exposed to left ureteral obstruction (UO), which continued for 24 hours, and then was released. 2 days after release of UO or at the corresponding time point in the non-UO sham-operated mice, the left kidneys were harvested and subjected to immunoblot analyses of HIF-2α and co-detection of TBP as a loading control

    Nuclear extracts were isolated from harvested whole kidneys using NE-PER Nuclear and Cytoplasmic Extraction Reagents supplemented with Complete Protease Inhibitor Cocktail Tablets. Nuclear protein fractions were electrophoresed on 10% SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. Membranes were blocked with LI-COR blocking buffer (LI-COR Biosciences, Lincoln, NE). Membranes were then incubated with the same blocking solution containing rabbit polyclonal primary antibodies against HIF-2α (1:500, ab199). After washing, membranes were incubated at room temperature for 1 h in TBS/0.05% Tween 20 buffer with the IRDye800 secondary antibodies (1:10000) and then washed again in TBS/0.05% Tween 20 for 3 times. The blot was visualized using an Odyssey infrared imaging system. All values were normalized to a loading control TATA binding protein (TBP, 1:2000, ab818, Abcam) and expressed as fold increase relative to control.

  • ICC/IF image of ab199 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab199, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • All lanes : Anti-HIF-2-alpha antibody - ChIP Grade (ab199) at 1/500 dilution

    Lane 1 : 20ug of whole cell lysate from PC12 cells.
    Lane 2 : 20ug of whole cell lysate from PC12 cells exposed to hypoxic conditions (12 hours at 3% oxygen).
    Lane 3 : 20ug of whole cell lysate from PC12 cells exposed to hypoxic conditions (12 hours at 3% oxygen) and incubated with antisense HIF2 alpha oligonucleotides 72 hours prior to treatment.

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 100 kDa
    Observed band size: 96 kDa (why is the actual band size different from the predicted?)



    The blots were probed with anti-alpha tubulin as a loading control.

文献

This product has been referenced in:
  • Serocki M  et al. miRNAs regulate the HIF switch during hypoxia: a novel therapeutic target. Angiogenesis 21:183-202 (2018). WB ; Human . Read more (PubMed: 29383635) »
  • Rodrigues P  et al. NF-?B-Dependent Lymphoid Enhancer Co-option Promotes Renal Carcinoma Metastasis. Cancer Discov 8:850-865 (2018). ChIP . Read more (PubMed: 29875134) »

See all 52 Publications for this product

客户评价及客户问答

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Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing (10% SDS PAGE)
Sample
Mouse Tissue lysate - whole (Mouse brain)
Specification
Mouse brain
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

提交于 Jun 02 2014

Application
Western blot
Sample
Human Tissue lysate - whole (Heart, Left Ventricle)
Loading amount
40 µg
Specification
Heart, Left Ventricle
Gel Running Conditions
Reduced Denaturing (4-12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Dawn Bowles

Verified customer

提交于 Jan 10 2013

Application
ChIP
Sample
Human Cell lysate - nuclear (Kidney (Clear Cell Renal Carcinoma))
Negative control
Parental clear cell renal carcinoma cells (786-O).
Specification
Kidney (Clear Cell Renal Carcinoma)
Detection step
Other
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% Formaldehyde
Positive control
Metastatic clear cell renal carcinoma cells (M1A).
Username

Paulo Rodrigues

Verified customer

提交于 Aug 17 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HUVEC)
Loading amount
20 µg
Specification
HUVEC
Treatment
Hypoxia 4 h
Gel Running Conditions
Reduced Denaturing (10 %)
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Username

Abcam user community

Verified customer

提交于 Mar 22 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (293 cells transfected with human HIF-2alpha expres)
Loading amount
100000 cells
Specification
293 cells transfected with human HIF-2alpha expres
Treatment
Cells were incubated in hypoxic condition (1% Oxygen) for 24hrs
Gel Running Conditions
Non-reduced Denaturing (10%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

提交于 Sep 04 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (adrenal)
Specification
adrenal
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate pH6
Blocking step
Serum as blocking agent for 10 minute(s) · Concentration: 10% · Temperature: 25°C
Username

Abcam user community

Verified customer

提交于 May 06 2008

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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