Name: Anti-NADPH oxidase 4 antibody [UOTR1B492]
Brand: RabMAb
SKU: ab109225
Rating: 5 (2 reviews)

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

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [UOTR1B492] to NADPH oxidase 4
Suitable for: Flow Cyt (Intra), ICC/IF, WB, IP, IHC-P
Reacts with: Human

Alexa Fluor® 488 Alexa Fluor® 647 Carrier Free HRP

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: Flow Cyt (Intra), ICC/IF, WB, IP, IHC-Pmore details
Species reactivity

Reacts with: Human
Predicted to work with: Rat, Dog
Immunogen

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
(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.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Store at -20°C. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 40% Glycerol, 59% PBS, 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

UOTR1B492
Isotype

IgG
Research areas

Alternative Versions
Compatible Secondaries
Conjugation kits
- PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
- APC Conjugation Kit - Lightning-Link® (ab201807)
Immunizing Peptide (Blocking)
- NADPH oxidase 4 peptide (ab179799)
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
Recombinant Protein
- Recombinant Human NADPH oxidase 4 protein (ab112414)
Related Products
- (-)-Cannabidiol, Natural cannabinoid (ab120448)

The Abpromise guarantee

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
Flow Cyt (Intra)		1/280. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
ICC/IF		1/200.
WB	(1)	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)	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.

Notes
Flow Cyt (Intra) 1/280. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
ICC/IF 1/200.
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.

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.
Target information above from: UniProt accession Q9NPH5 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 50507 Human
- Entrez Gene: 85431 Rat
- Omim: 605261 Human
- SwissProt: Q9NPH5 Human
- SwissProt: Q924V1 Rat
- Unigene: 371036 Human
- Unigene: 14744 Rat
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

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)

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)

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)

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)

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

Intracellular 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 Fluorr^® 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)

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)

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)

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)

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

Flow cytometry protocols
Immunoprecipitation protocols
Immunohistochemistry protocols
Immunocytochemistry & immunofluorescence protocols
Western blot protocols

Click here to view the general protocols

SDS download

Country/Region

Language
Datasheet download

Download

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

ab109225 has been referenced in 73 publications.

Zhao HY et al. L-carnitine treatment attenuates renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction. Korean J Intern Med 36:S180-S195 (2021). PubMed: 32942841
Sun CY et al. Indoxyl sulfate caused behavioral abnormality and neurodegeneration in mice with unilateral nephrectomy. Aging (Albany NY) 13:6681-6701 (2021). PubMed: 33621199
Liu N et al. NADPH and Mito-Apocynin Treatment Protects Against KA-Induced Excitotoxic Injury Through Autophagy Pathway. Front Cell Dev Biol 9:612554 (2021). PubMed: 33644049
Cao JY et al. Autophagosome protects proximal tubular cells from aldosterone-induced senescence through improving oxidative stress. Ren Fail 43:556-565 (2021). PubMed: 33757397
Zhou H et al. ASMq protects against early burn wound progression in rats by alleviating oxidative stress and secondary mitochondria-associated apoptosis via the Erk/p90RSK/Bad pathway. Mol Med Rep 23:N/A (2021). PubMed: 33760179

View all Publications for this product

Customer reviews and Q&As

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1-10 of 13 Abreviews or Q&A

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

Fixative

Formaldehyde

Abcam user community

Verified customer

Submitted Sep 09 2013

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

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

Abcam community

Verified customer

Asked on Jan 23 2013

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.

Abcam Scientific Support

Answered on Jan 23 2013

Question

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

Abcam community

Verified customer

Asked on Dec 17 2012

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.

Abcam Scientific Support

Answered on Dec 17 2012

Question

What is the concentration of product ab109225?

Abcam community

Verified customer

Asked on Oct 19 2012

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.

Abcam Scientific Support

Answered on Oct 19 2012

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.

Abcam community

Verified customer

Asked on Oct 11 2012

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.

Abcam Scientific Support

Answered on Oct 11 2012

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.

Abcam community

Verified customer

Asked on Oct 11 2012

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.

Abcam Scientific Support

Answered on Oct 11 2012

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,

Abcam community

Verified customer

Asked on Sep 25 2012

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!
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Abcam Scientific Support

Answered on Sep 25 2012

Question

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

Abcam community

Verified customer

Asked on Aug 22 2012

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.

Abcam Scientific Support

Answered on Aug 22 2012

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

Abcam community

Verified customer

Asked on Jun 11 2012

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

Abcam Scientific Support

Answered on Jun 11 2012

1-10 of 13 Abreviews or Q&A

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Key features and details

Related conjugates and formulations

Overview

Product name

Description

Host species

Tested applications

Species reactivity

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Storage buffer

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

Compatible Secondaries

Conjugation kits

Immunizing Peptide (Blocking)

Isotype control

Recombinant Protein

Related Products

Applications

The Abpromise guarantee

Target

Function

Tissue specificity

Sequence similarities

Developmental stage

Post-translationalmodifications

Cellular localization

Database links

Alternative names

Images

Protocols

Datasheets and documents

SDS download

Datasheet download

References (73)

Customer reviews and Q&As

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

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

Post-translational
modifications