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NADPH oxidase 4/NOX4 Recombinant Rabbit Monoclonal Antibody [SY0214]

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Specification

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Catalog# ET1607-4

NADPH oxidase 4/NOX4 Recombinant Rabbit Monoclonal Antibody [SY0214]

Application

  • WB

  • IF-Cell

  • IHC-P

  • IP

  • FC

Reactivity

  • Human

  • Rat

BSA and Azide free
Predicted reactivity

  • Mouse

Conjugation

  • unconjugated

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

Overview

Product Name

NADPH oxidase 4/NOX4 Recombinant Rabbit Monoclonal Antibody [SY0214]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Recombinant protein within Human NOX4 aa 460-578 / 578.

Species Reactivity

Human, Rat (Predicted: Mouse)

Predicted species support after-sales service

Validated Applications

WB, IF-Cell, IHC-P, IP, FC

Target Molecular Weight

Predicted band size: 67 kDa

Positive Control

SH-SY5Y cell lysate, U-2 OS cell lysate, SH-SY5Y, human lung tissue, human kidney tissue, rat stomach tissue, rat kidney tissue.

Conjugation

unconjugated

Clone Number

SY0214

RRID

AB_3069764

Reactivity Data

Tested Tested Tested
Verified (internally validated)
Published Tested
Reported in literature (not internally validated)
Predicted Tested
Predicted reactive (based on sequence homology)
Not recommended Tested
Not recommended (failed internal validation)
WB FC IF-Cell IP
human
Tested Tested
Tested Tested
Tested Tested
Tested Tested
mouse
Not Recommended
 
 
 
rat
Not Recommended
 
 
 

Product Features

Form

Liquid

Concentration

1 mg/mL.(The concentration of this product may be batch-dependent)

Storage Instructions

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

Storage Buffer

1*PBS (pH7.4), 0.1% BSA, 40% Glycerol, 0.2% Proclean 950.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:2,000

  • IF-Cell

  • 1:100

  • IHC-P

  • 1:200-1:500

  • IP

  • 1-2μg/sample

  • FC

  • 1:1,000

Target

Function

The superoxide-generating NADPH oxidase includes a membrane-bound flavocytochrome containing two subunits, gp91-phox and p22-phox, and the cytosolic proteins p47-phox and p67-phox. During activation of the NADPH oxidase, p47-phox and p67-phox migrate to the plasma membrane where they associate with the flavocytochrome, cytochrome b558, to form the active enzyme complex. The p22 and gp91-phox subunits also function as surface O2 sensors that initiate cellular signaling in response to hypoxic conditions. Nox4 (also known as Renox) is a renal gp91-phox homolog highly expressed at the site of erythropoietin production in the proximal convoluted tubule epithelial cells of the renal cortex. Nox4 is also expressed in fetal tissues, placenta, glioblastoma and vascular cells. Like gp91-phox, the enzymatic activity of Nox4 produces superoxide anions. In vascular cells, the addition of Angiotensin II increases Nox4 expression, which suggests a role for Nox4 in vascular oxidative stress response. The gene encoding human Nox4 maps to chromosome 11q14.2-q21.

Background References

1. Yu P et al. A dual role of transient receptor potential melastatin 2 channel in cytotoxicity induced by silica nanoparticles. Sci Rep 5:18171 (2015).

2. Dvash E et al. Leukotriene C4 is the major trigger of stress-induced oxidative DNA damage. Nat Commun 6:10112 (2015).

Tissue Specificity

Expressed by distal tubular cells in kidney cortex and in endothelial cells (at protein level). Widely expressed. Strongly expressed in kidney and to a lower extent in heart, adipocytes, hepatoma, endothelial cells, skeletal muscle, brain, several brain tumor cell lines and airway epithelial cells.

Post-translational Modification

Isoform 3 and isoform 4 are N-glycosylated. Isoform 4 glycosylation is required for its proper function.

Subcellular Location

Cell membrane, Nucleus, Endoplasmic reticulum membrane.

UNIPROT

SwissProt: Q9NPH5 Human

SwissProt: Q924V1 Rat

Synonyms

Kidney oxidase-1 antibody

Kidney superoxide-producing NADPH oxidase antibody

KOX 1 antibody

KOX antibody

Kox-1 antibody

NADPH antibody

NADPH oxidase 4 antibody

Nox4 antibody

NOX4_HUMAN antibody

Renal NAD(P)H-oxidase antibody

Expand

Images

  • Western blot analysis of NADPH oxidase 4/NOX4 on different lysates with Rabbit anti-NADPH oxidase 4/NOX4 antibody (<a href="/products/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</a>) at 1/2,000 dilution.<br /><br />Lane 1: SH-SY5Y cell lysate<br />Lane 2: U-2 OS cell lysate<br /><br />Lysates/proteins at 15 µg/Lane.<br /><br />Predicted band size: 67 kDa<br />Observed band size: 67 kDa<br /><br />Exposure time: 3 minutes;<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/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</a>) at 1/2,000 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 NADPH oxidase 4/NOX4 on different lysates with Rabbit anti-NADPH oxidase 4/NOX4 antibody (ET1607-4) at 1/2,000 dilution.

    Lane 1: SH-SY5Y cell lysate
    Lane 2: U-2 OS cell lysate

    Lysates/proteins at 15 µg/Lane.

    Predicted band size: 67 kDa
    Observed band size: 67 kDa

    Exposure time: 3 minutes;

    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 (ET1607-4) at 1/2,000 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.

  • <span style="font-weight: bold;">☑ Knockdown (KD)</span><br /><br />Western blot analysis of NOX4 with anti-NOX4 antibody [SY0214] (<a href="/products/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</a>) at 1/1,000 dilution.<br /><br />Lane 1: Wild-type SH-SY5Y whole cell lysate (20 µg).<br />Lane 2/3: NOX4 fragment 1 knockdown SH-SY5Y whole cell lysate (20 µg).<br />Lane 4/5: NOX4 fragment 2 knockdown SH-SY5Y whole cell lysate (20 µg).<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</a>, 1/1,000) and Loading control antibody(Rabbit anti-HSP90, <a href="/products/ET1605-56" style="font-weight: bold;text-decoration: underline;">ET1605-56</a>, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG-HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/200,000 dilution was used for 1 hour at room temperature.

    ☑ Knockdown (KD)

    Western blot analysis of NOX4 with anti-NOX4 antibody [SY0214] (ET1607-4) at 1/1,000 dilution.

    Lane 1: Wild-type SH-SY5Y whole cell lysate (20 µg).
    Lane 2/3: NOX4 fragment 1 knockdown SH-SY5Y whole cell lysate (20 µg).
    Lane 4/5: NOX4 fragment 2 knockdown SH-SY5Y whole cell lysate (20 µg).

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1607-4, 1/1,000) and Loading control antibody(Rabbit anti-HSP90, ET1605-56, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG-HRP Secondary Antibody (HA1001) at 1/200,000 dilution was used for 1 hour at room temperature.

  • NADPH oxidase 4/NOX4 was immunoprecipitated in 0.2mg SH-SY5Y cell lysate with <a href="/products/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</a> at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using <a href="/products/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</a> at 1/1,000 dilution. Anti-Rabbit IgG for IP, AlpSdAbs&reg; VHH(HRP) (<a href="/products/025-100-005" style="font-weight: bold;text-decoration: underline;">025-100-005</a>) at 1/5,000 dilution was used for 1 hour at room temperature.<br /><br />Lane 1: SH-SY5Y cell lysate (input)<br />Lane 2: Rabbit IgG instead of <a href="/products/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</a> in SH-SY5Y cell lysate<br />Lane 3: <a href="/products/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</a> IP in SH-SY5Y cell lysate<br /><br />Blocking/Dilution buffer: 5% NFDM/TBST<br />Exposure time: 24 seconds; ECL: <a href="/products/K1801" style="font-weight: bold;text-decoration: underline;">K1801</a>

    NADPH oxidase 4/NOX4 was immunoprecipitated in 0.2mg SH-SY5Y cell lysate with ET1607-4 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1607-4 at 1/1,000 dilution. Anti-Rabbit IgG for IP, AlpSdAbs® VHH(HRP) (025-100-005) at 1/5,000 dilution was used for 1 hour at room temperature.

    Lane 1: SH-SY5Y cell lysate (input)
    Lane 2: Rabbit IgG instead of ET1607-4 in SH-SY5Y cell lysate
    Lane 3: ET1607-4 IP in SH-SY5Y cell lysate

    Blocking/Dilution buffer: 5% NFDM/TBST
    Exposure time: 24 seconds; ECL: K1801

  • Immunocytochemistry analysis of SH-SY5Y cells labeling NADPH oxidase 4/NOX4 with Rabbit anti-NADPH oxidase 4/NOX4 antibody (<a href="/products/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</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-NADPH oxidase 4/NOX4 antibody (<a href="/products/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</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 SH-SY5Y cells labeling NADPH oxidase 4/NOX4 with Rabbit anti-NADPH oxidase 4/NOX4 antibody (ET1607-4) 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-NADPH oxidase 4/NOX4 antibody (ET1607-4) 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 SH-SY5Y cells labeling NADPH oxidase 4/NOX4.<br /><br />Cells were fixed and permeabilized. Then stained with the primary antibody (<a href="/products/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</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 SH-SY5Y cells labeling NADPH oxidase 4/NOX4.

    Cells were fixed and permeabilized. Then stained with the primary antibody (ET1607-4, 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).

  • Immunohistochemical analysis of paraffin-embedded human kidney tissue using anti-NADPH oxidase 4/NOX4 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes.The tissues were blocked in 1% BSA for 30 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</a>, 1/50) for 30 minutes 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 kidney tissue using anti-NADPH oxidase 4/NOX4 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes.The tissues were blocked in 1% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1607-4, 1/50) for 30 minutes 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 rat kidney tissue using anti-NADPH oxidase 4/NOX4 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes.The tissues were blocked in 1% BSA for 30 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1607-4" style="font-weight: bold;text-decoration: underline;">ET1607-4</a>, 1/200) for 30 minutes 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 rat kidney tissue using anti-NADPH oxidase 4/NOX4 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes.The tissues were blocked in 1% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1607-4, 1/200) for 30 minutes 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.

Citation

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

Products with the same target and pathway

metafield image

NADPH oxidase 4/NOX4 Recombinant Rabbit Monoclonal Antibody [SY0214] - BSA and Azide free

Application: WB,IF-Cell,IHC-P,IP,FC

Reactivity: Human,Rat,Mouse

Conjugate: unconjugated