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HIF-2 alpha Recombinant Rabbit Monoclonal Antibody [JB24-42]

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Specification

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Catalog# ET7107-32

HIF-2 alpha Recombinant Rabbit Monoclonal Antibody [JB24-42]

Application

  • WB

  • IHC-P

  • FC

  • IF-Cell

Reactivity

  • Human

  • Mouse

  • Rat

BSA and Azide free
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

HIF-2 alpha Recombinant Rabbit Monoclonal Antibody [JB24-42]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic peptide within Human HIF-2 alpha aa 61-110 / 870.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IHC-P, FC, IF-Cell

Molecular Weight

Predicted band size: 96 kDa

Positive Control

SiHa cell lysate, Mouse lung tissue lysate, human breast tissue, human placenta tissue, mouse colon tissue, rat lung tissue, SiHa.

Conjugation

unconjugated

Clone Number

JB24-42

RRID

AB_2938782

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:500-1:2,000

  • IHC-P

  • 1:50-1:1,000

  • FC

  • 1:1,000

  • IF-Cell

  • 1:100

Target

Function

Endothelial PAS domain-containing protein 1 (EPAS1, also known as hypoxia-inducible factor-2alpha (HIF-2α)) is a protein that is encoded by the EPAS1 gene in mammals. It is a type of hypoxia-inducible factor, a group of transcription factors involved in the physiological response to oxygen concentration. The gene is active under hypoxic conditions. It is also important in the development of the heart, and for maintaining the catecholamine balance required for protection of the heart. Mutation often leads to neuroendocrine tumors. The EPAS1 gene encodes one subunit of a transcription factor involved in the induction of genes regulated by oxygen, and which is induced as oxygen concentration falls (hypoxia). The protein contains a basic helix-loop-helix protein dimerization domain as well as a domain found in signal transduction proteins which respond to oxygen levels. EPAS1 is involved in the development of the embryonic heart and is expressed in endothelial cells that line the walls of blood vessels in the umbilical cord. EPAS1 is also essential for the maintenance of catecholamine homeostasis and protection against heart failure during early embryonic development. Catecholamines regulated by EPAS1 include epinephrine and norepinephrine. It is critical that the production of catecholamines remain in homeostatic conditions so that both the delicate fetal heart and the adult heart do not overexert themselves and induce heart failure. Catecholamine production in the embryo is related to control of cardiac output by increasing the fetal heart rate.

Background References

1. Ema M et al. Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300. EMBO J 18:1905-1914 (1999).

2. Furlow P W et al. Erythrocytosis-associated HIF-2alpha mutations demonstrate a critical role for residues C-terminal to the hydroxylacceptor proline. J Biol Chem 284:9050-9058 (2009).

Tissue Specificity

Expressed in most tissues, with highest levels in placenta, lung and heart. Selectively expressed in endothelial cells.

Post-translational Modification

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 (By similarity).; 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.

Subcellular Location

Nucleus, Nucleus speckle.

UNIPROT

SwissProt: Q99814 Human

SwissProt: P97481 Mouse

SwissProt: Q9JHS1 Rat

Synonyms

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

Expand

Images

  • Western blot analysis of HIF-2 alpha on different lysates with Rabbit anti-HIF-2 alpha antibody (<a href="/products/ET7107-32" style="font-weight: bold;text-decoration: underline;">ET7107-32</a>) at 1/1,000 dilution.<br /><br />Lane 1: SiHa cell lysate (negative) (10 µg/Lane)<br />Lane 2: Mouse lung tissue lysate (20 µg/Lane)<br /><br />Predicted band size: 96 kDa<br />Observed band size: 110 kDa<br /><br />Exposure time: 1 minute; ECL: K1801;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET7107-32" style="font-weight: bold;text-decoration: underline;">ET7107-32</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of HIF-2 alpha on different lysates with Rabbit anti-HIF-2 alpha antibody (ET7107-32) at 1/1,000 dilution.

    Lane 1: SiHa cell lysate (negative) (10 µg/Lane)
    Lane 2: Mouse lung tissue lysate (20 µg/Lane)

    Predicted band size: 96 kDa
    Observed band size: 110 kDa

    Exposure time: 1 minute; ECL: K1801;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET7107-32) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • Immunohistochemical analysis of paraffin-embedded human breast tissue with Rabbit anti-HIF-2 alpha antibody (<a href="/products/ET7107-32" style="font-weight: bold;text-decoration: underline;">ET7107-32</a>) at 1/1,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET7107-32" style="font-weight: bold;text-decoration: underline;">ET7107-32</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human breast tissue with Rabbit anti-HIF-2 alpha antibody (ET7107-32) at 1/1,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET7107-32) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded human placenta tissue using anti-HIF-2 alpha antibody. Counter stained with hematoxylin.

    Immunohistochemical analysis of paraffin-embedded human placenta tissue using anti-HIF-2 alpha antibody. Counter stained with hematoxylin.

  • Immunohistochemical analysis of paraffin-embedded mouse colon tissue using anti-HIF-2 alpha antibody. Counter stained with hematoxylin.

    Immunohistochemical analysis of paraffin-embedded mouse colon tissue using anti-HIF-2 alpha antibody. Counter stained with hematoxylin.

  • Immunohistochemical analysis of paraffin-embedded rat lung tissue using anti-HIF-2 alpha antibody. Counter stained with hematoxylin.

    Immunohistochemical analysis of paraffin-embedded rat lung tissue using anti-HIF-2 alpha antibody. Counter stained with hematoxylin.

  • Immunocytochemistry analysis of SiHa cells labeling HIF-2 alpha with Rabbit anti-HIF-2 alpha antibody (<a href="/products/ET7107-32" style="font-weight: bold;text-decoration: underline;">ET7107-32</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HIF-2 alpha antibody (<a href="/products/ET7107-32" style="font-weight: bold;text-decoration: underline;">ET7107-32</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of SiHa cells labeling HIF-2 alpha with Rabbit anti-HIF-2 alpha antibody (ET7107-32) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-HIF-2 alpha antibody (ET7107-32) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Flow cytometric analysis of SiHa cells labeling HIF-2 alpha.<br /><br />Cells were fixed and permeabilized. Then stained with the primary antibody (<a href="/products/ET7107-32" style="font-weight: bold;text-decoration: underline;">ET7107-32</a>, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor&trade; 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (<a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    Flow cytometric analysis of SiHa cells labeling HIF-2 alpha.

    Cells were fixed and permeabilized. Then stained with the primary antibody (ET7107-32, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

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