Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)

Overview

  • Product nameAnti-NADPH oxidase 4 antibody [UOTR1B492]
    See all NADPH oxidase 4 primary antibodies
  • Description
    Rabbit monoclonal [UOTR1B492] to NADPH oxidase 4
  • SpecificityFor detection of NADPH oxidase 4 in mouse samples we recommend ab133303.
  • Tested applicationsSuitable for: ICC/IF, Flow Cyt, WB, IP, IHC-Pmore details
  • Species reactivity
    Reacts with: Rat, Dog, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human NADPH oxidase 4 aa 500 to the C-terminus.
    (Peptide available as ab179799)

  • Positive control
    • Fetal kidney, U87-MG, 293T, and JAR lysates Human kidney tissue
  • General notes

    This product is a recombinant rabbit monoclonal antibody.

     

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated ‘PUR’ on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

    Alternative versions available:

    Anti-NADPH oxidase 4 antibody (Alexa Fluor® 488) [UOTR1B492] (ab195300)

    Anti-NADPH oxidase 4 antibody (Alexa Fluor® 647) [UOTR1B492] (ab195301)

    Anti-NADPH oxidase 4 antibody (HRP) [UOTR1B492] (ab195524)

Properties

Applications

Our Abpromise guarantee covers the use of ab109225 in the following tested applications.

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

Application Abreviews Notes
ICC/IF 1/200.
Flow Cyt 1/280.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB 1/2000. Detects a band of approximately 63 kDa (predicted molecular weight: 67 kDa).Can be blocked with NADPH oxidase 4 peptide (ab179799).
IP 1/30.
IHC-P 1/250 - 1/500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).

For unpurified, use 1/100 - 1/250.

Target

  • FunctionConstitutive NADPH oxidase which generates superoxide intracellularly upon formation of a complex with CYBA/p22phox. Regulates signaling cascades probably through phosphatases inhibition. May function as an oxygen sensor regulating the KCNK3/TASK-1 potassium channel and HIF1A activity. May regulate insulin signaling cascade. May play a role in apoptosis, bone resorption and lipolysaccharide-mediated activation of NFKB. May produce superoxide in the nucleus and play a role in regulating gene expression upon cell stimulation. Isoform 3 is not functional. Isoform 4 displays an increased activity. Isoform 5 and isoform 6 display reduced activity.
  • Tissue specificityExpressed 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.
  • Sequence similaritiesContains 1 FAD-binding FR-type domain.
    Contains 1 ferric oxidoreductase domain.
  • Developmental stageExpressed in fetal kidney and fetal liver.
  • Post-translational
    modifications
    Isoform 3 and isoform 4 are N-glycosylated. Isoform 4 glycosylation is required for its proper function.
  • Cellular localizationEndoplasmic reticulum membrane. Cell membrane. Cell junction > focal adhesion. Nucleus. May localize to plasma membrane and focal adhesions. According to PubMed:15927447, may also localize to the nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • Kidney oxidase 1 antibody
    • Kidney oxidase-1 antibody
    • Kidney superoxide producing NADPH oxidase antibody
    • Kidney superoxide-producing NADPH oxidase antibody
    • KOX 1 antibody
    • KOX antibody
    • Kox-1 antibody
    • KOX1 antibody
    • NADPH antibody
    • NADPH oxidase 4 antibody
    • Nox4 antibody
    • NOX4_HUMAN antibody
    • Renal NAD(P)H oxidase antibody
    • Renal NAD(P)H-oxidase antibody
    • RENOX antibody
    see all

Anti-NADPH oxidase 4 antibody [UOTR1B492] images

  • Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225) at 1/10000 dilution (purified) + JAR cell lysate at 10 µg

    Secondary
    HRP goat anti-rabbit (H+L) at 1/1000 dilution

    Predicted band size : 67 kDa
    Observed band size : 63 kDa (why is the actual band size different from the predicted?)

    Blocking buffer: 5% NFDM/TBST

    Dilution buffer: 5% NFDM/TBST

     

  • Immunohistochemical staining of paraffin embedded human stomach with purified ab109225 at a dilution of 1/500. A HRP goat anti-rabbit (ab97051) was used as the secondary antibody at a dilution of 1/500 and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

  • Immunofluorescent staining of HeLa cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab109225 at a dilution of 1/200. An Alexa Fluor® 488 goat anti-rabbit antibody was used as the secondary at a dilution of 1/500 and the cells were counter stained with DAPI. The negative control is shown in the bottom right hand panel - for the negative control, Alex Fluor® 594 goat anti-mouse was used at a dilution of 1/500.

  • Flow Cytometry analysis of U87-MG (human glioblastoma) cells labeling NADPH oxidase 4 with purified ab109225 at 1/280 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

  • Immunohistochemical staining of paraffin embedded mouse cerebral cortex with purified ab109225 at a dilution of 1/500. A HRP goat anti-rabbit (ab97051) was used as the secondary antibody at a dilution of 1/500 and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

  • Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225) at 1/2000 dilution (purified) + Human fetal kidney at 10 µg

    Secondary
    HRP anti-rabbit, specific to the non reducded form of IgG at 1/1000 dilution

    Predicted band size : 67 kDa
    Observed band size : 63 kDa (why is the actual band size different from the predicted?)

    Blocking buffer: 5% NFDM/TBST

    Dilution buffer: 5% NFDM/TBST

  • ab109225 (purified) at 1/30 immunoprecipitating NADPH oxidase 4 in HEK293. For western blotting, a HRP-conjugated anti-rabbit antibody was used as the secondary antibody (1/1000).

    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

  • All lanes : Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225) at 1/1000 dilution (unpurified)

    Lane 1 : Fetal kidney lysate
    Lane 2 : U87-MG lysate
    Lane 3 : 293T lysate
    Lane 4 : JAR lysates

    Lysates/proteins at 10 µg per lane.

    Secondary
    Standard HRP labelled goat anti-rabbit at 1/2000 dilution

    Predicted band size : 67 kDa
    Observed band size : 63 kDa (why is the actual band size different from the predicted?)
  • Immunohistochemical analysis of paraffin-embedded Human kidney tissue using unpurified ab109225.

  • ICC/IF image of unpurified ab109255 stained HeLa cells. The cells were 4% formaldehyde 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 (ab109225, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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.

  • Unpurified ab109225 staining Nox4 in HeLa cells treated with (-)-cannabidiol (ab120448), by ICC/IF. Increase in Nox4 expression correlates with increased concentration of (-)-cannabidiol, as described in literature.
    The cells were incubated at 37°C for 6h in media containing different concentrations of ab120448 ((-)-cannabidio) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab109225 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody.

References for Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)

This product has been referenced in:
  • López-Álvarez GS  et al. Gene silencing of Nox4 by CpG island methylation during hepatocarcinogenesis in rats. Biol Open 6:59-70 (2017). ICC/IF ; Rat . Read more (PubMed: 27895046) »
  • Holley CL  et al. Cytosolic accumulation of small nucleolar RNAs (snoRNAs) is dynamically regulated by NADPH oxidase. J Biol Chem 290:11741-8 (2015). WB ; Rat . Read more (PubMed: 25792744) »

See all 14 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Power block as blocking agent for 5 minute(s) · Concentration: 10% · Temperature: 21°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: citrate pH 6.0
Sample Dog Tissue sections (Kidney)
Specification Kidney
Permeabilization No
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Sep 09 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (Glioblastoma primary cells)
Loading amount 25 µg
Specification Glioblastoma primary cells
Gel Running Conditions Reduced Denaturing (12.5%)
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted May 08 2013

Thank you once again for your enquiries,.

I can confirm you can use the same blocking peptide for ab109225:
ab155071 Nox 4 peptide

I hope this will be helpful. If you have any further questions, please do not hesitate to cont...

Read More

This immunogen ss intracellular. It has 54% identity with NOX1, no significant sequence similarity with NOX2, 47% identity with NOX3, and no homology with NOX5. In order to aid your research we are happy to provide this information to you. However, ...

Read More

Thank you for your inquiry regarding ab109225.


I can confirm that teh recent batch is supplied at 2.0000 mg/ml.


If you need any further assistance in the future, please do not hesitate to contact me.

Thank you for your reply.

I am sorry that you sre still not satisfied with the specificity of these 4 NOX4 antibodies and as requested I have processed a refund.

Your credit note ID is 22995.

I am sorry that this antibod...

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Thank you for contacting Abcam.



After having talked with the lab, we agree that your data looks good. I see that there is a band near 63 KDa (the expected molecular weight) with all of the antibodies and even in couple of your blots ...

Read More

Thank you for contacting us.

This product was developed by one of our collaborator university laboratory. They are having difficulties in product so the product is unpublished momentarily.

The product ab109225 is a rabbit monoclonal a...

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Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has cau...

Read More

Thank you for contacting us.
I am sorry to hear that the antibody is not working as stated on the datasheet.

As we discussed over the phone, I would suggest the following:
1) load more protein (e.g. 30- 50 ug) use more antibody
Read More

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