Recombinant
RabMAb

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

Reviews (2)Q&A (11)References (43)

Overview

  • Product name

    Anti-NADPH oxidase 4 antibody [UOTR1B492]
    See all NADPH oxidase 4 primary antibodies

  • Description

    Rabbit monoclonal [UOTR1B492] to NADPH oxidase 4

  • Host species

    Rabbit

  • Tested applications

    Suitable 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 monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    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.

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 IHC antigen retrieval protocols.

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

Target

  • Function

    Constitutive 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 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.

  • Sequence similarities

    Contains 1 FAD-binding FR-type domain.
    Contains 1 ferric oxidoreductase domain.

  • Developmental stage

    Expressed 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 localization

    Endoplasmic 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

    Images

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

       

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)

      Immunohistochemical analysis of paraffin-embedded Human kidney tissue using unpurified ab109225.

      Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

    • Immunocytochemistry/ Immunofluorescence - Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)
      Immunocytochemistry/ Immunofluorescence - Anti-NADPH oxidase 4 antibody [UOTR1B492] (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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)

      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.

    • Immunocytochemistry/ Immunofluorescence - Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)
      Immunocytochemistry/ Immunofluorescence - Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)

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

      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.

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

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

      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.

    • Western blot - Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)
      Western blot - Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)
      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
      All lanes : 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?

    • Immunocytochemistry/ Immunofluorescence - Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)
      Immunocytochemistry/ Immunofluorescence - Anti-NADPH oxidase 4 antibody [UOTR1B492] (ab109225)

      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

    This product has been referenced in:

    • Wu QQ  et al. Downregulated NOX4 underlies a novel inhibitory role of microRNA-137 in prostate cancer. J Cell Biochem N/A:N/A (2019). Read more (PubMed: 30637800) »
    • Jiang Y  et al. Cryptotanshinone Ameliorates Radiation-Induced Lung Injury in Rats. Evid Based Complement Alternat Med 2019:1908416 (2019). Read more (PubMed: 30915142) »
    See all 43 Publications for this product

    Publishing research using ab109225? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-10 of 13 Abreviews or Q&A

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-NADPH oxidase 4 antibody [UOTR1B492]

     Good
    Abreviews
    abreview image
    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
    Read More

    Abcam user community

    Verified customer

    Submitted Sep 09 2013

    Western blot abreview for Anti-NADPH oxidase 4 antibody [UOTR1B492]

     Excellent
    Abreviews
    abreview image
    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
    Read More

    Abcam user community

    Verified customer

    Submitted May 08 2013

    Question

    Thanks very much Kate. Much appreciated. I will order as soon as you let me know it is there.

    Best wishes

    Read More
    Answer

    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 contact us.

    Read More

    Question

    Customer kindly contacted us to inquire about the epitope sequence of this product.

    Read More
    Answer

    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, this immunogen sequence is proprietary (commercially sensitive and confidential). Therefore, please do not share or publish this information. It is strictly for your own use.

    Read More

    Question

    What is the concentration of product ab109225?

    Read More
    Answer

    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.

    Read More

    Question

    If only the monoclonal antibodies behaved as this paper suggests. After looking at the paper, the supplimental data and figure 1 clearly demonstrates that with HEK293 cells alone there should only be a faint band observed (which is my vector control) and the over expression of NOX4 gives a clear, dark band. In my hands with the same system (where I can show the NOX4 is overexpressed both by increased p22 due to stabilization through NOX4 interaction and by Myc tag - as well as higher H2O2 production against the vector control which I haven't shown you) I am not seeing this result - the vector control should show a faint band relative to the overexpressed NOX4 HEK293 cells. I can't verify what the authors of this paper have done/published, I only know that my data does not support these results. I would still greatly appreciate a refund for these antibodies (order#1152887), and urge caution with these antibodies as I am still concerned they are not truly recognizing NOX4.

    Read More
    Answer

    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 antibody did not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.

    Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.

    The credit note ID is for your reference only and does not automatically guarantee the credit.

    I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

    Read More

    Question

    To whom it may concern,
    I am emailing in regards to order #***placed with Abcam on ****, requesting 4 antibodies (ab109225, ab133303, ab60940 and ab61248) against the protein NOX4. Our research is in the NOX/DUOX protein family and their implication in cancer, and we have been searching for an antibody that recognizes the NOX4protein. Attached is the results of our western blots of an overexpression system for NOX4 (vector control vs 5' Myc tagged NOX4 overexpressed in HEK293) as well as a NOX4 clone (clone F), and SKMEL5 cells which have an albeit low RNA level of NOX4. The results of your antibodies (while the Myc tag control loaded at the same time, same day for all gels/westerns clearly shows overexpressed NOX4) do not demonstrate these antibodies are detecting NOX4 at all, as the 5' overexpression should clearly demonstrate a higher level of NOX4 than the vector control. I believe the antibody is recognizing an HEK protein, simply not NOX4 (plus the RNA levels of NOX4 in HEK are around 800-2000, even lower than SKMEL5, so really no band should be visible for the vector control lane). It is an acknowledged problem in the field that companies cannot create a good antibody to recognize this protein (see Babelova, A. et al Free Radic Biol Med. 2012, 15, 842-853, Figure 1D), we simply had hopes these antibodies might work. As we are unsatisfied with the results, I am requesting that the monies used for purchase be refunded to our account at the NIH.

    Read More
    Answer

    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 there looks to small increases in expression levels in your transfected lanes.


    Unfortunately, we do not have QC data that we can send to show specificity, however, two of these antibodies (ab109225 (3187-1) & ab133303 (#3174-1) ) were produced as part of a collaboration and they ran controls to verify that the antibody is specific to NOX4. I would refer you to the Results section of the following publication because they state that the antibody has been confirmed to be specific ("Using lysates from both Nox4 overexpressing cells and cells from Nox4 knockout mice, we confirmed the specificity of both the rabbit polyclonal (not shown) and monoclonal Nox4 antibodies"):



    Lee, C.F., Qiao, M. Schroeder, K., Zhao, Q. and Asmis, R., Nox4 is a novel inducible source of reactive oxygen species in monocytes and macrophages and mediates oxidized low density lipoprotein-induced macrophage death. ,Circ.Res. 106(9): 1489-97 (2010).



    If after looking at this paper, you still do not agree with the results, then please let me know and I will process a refund.



    Please let me know if you have any further questions.

    Read More

    Question

    We have a customer who is wondering the reason for the discontinuation of this antibody as it was the best antibody he could find for NOX4.

    He would also like to know if AB109225 is the same antibody but under a different catalogue number?

    Thanks for your help and hope to hear from you soon

    Kind regards,

    Read More
    Answer

    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 antibody against NOX4 and is a different product. I would suggest trying this product as alternative.

    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

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    Question

    Customer kindly contacted us as ab116534 did not work in WB on human cell line samples.

    Read More
    Answer

    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 caused. As requested, I have issued a free of charge replacement with the order number xxxxx. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.

    Read More

    Question

    WB with HUVEC
    no signal
    reduced-denaturing conditions
    used more than 10 ug
    Ab: 1/1000
    other protein detected, thus transfer works
    2nd: OK

    suggested: use more protein, use more antibody

    Read More
    Answer

    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
    2) use more primary antibody (e.g. 1/500)

    Please let me know if these tips are of help or not.
    If not, I'd be happy to discuss other options to resolve this problem if the antibody was purchased within the last 6 months.

    I wish you good luck and look forward to hear back from you.

    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

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    1-10 of 13 Abreviews or Q&A

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