Overview

  • Product name

    Anti-NOX2/gp91phox antibody
    See all NOX2/gp91phox primary antibodies
  • Description

    Rabbit polyclonal to NOX2/gp91phox
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, Flow Cyt, IHC-Fr, IHC-P, WBmore details
  • Species reactivity

    Reacts with: Mouse, Human
    Predicted to work with: Rabbit, Cow, Dog
  • Immunogen

    Synthetic peptide corresponding to Human NOX2/gp91phox aa 511-560 (C terminal).
    Sequence:

    YGRPNWDNEFKTIASQHPNTRIGVFLCGPEALAETLSKQSISNSESGPRG


    (Peptide available as ab111671)

  • Positive control

    • MCF7 cell lysate. THP-1 cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab80508 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 Use a concentration of 1 µg/ml.
Flow Cyt Use at an assay dependent concentration.
IHC-Fr Use a concentration of 5 µg/ml.
IHC-P Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 65 kDa (predicted molecular weight: 65 kDa).

Good results were obtained when blocked with 5% non-fat dry milk in 0.05% PBS-T.

As published in databases Human Protein Atlas, GeneCard and NCBI CYBB expression in heart is cell type specific therefore, we recommend including positive controls when using this antibody with heart samples.

Target

  • Function

    Critical component of the membrane-bound oxidase of phagocytes that generates superoxide. It is the terminal component of a respiratory chain that transfers single electrons from cytoplasmic NADPH across the plasma membrane to molecular oxygen on the exterior. Also functions as a voltage-gated proton channel that mediates the H(+) currents of resting phagocytes. It participates in the regulation of cellular pH and is blocked by zinc.
  • Involvement in disease

    Defects in CYBB are a cause of chronic granulomatous disease X-linked (XCGD) [MIM:306400]. Chronic granulomatous disease is a genetically heterogeneous disorder characterized by the inability of neutrophils and phagocytes to kill microbes that they have ingested. Patients suffer from life-threatening bacterial/fungal infections.
  • Sequence similarities

    Contains 1 FAD-binding FR-type domain.
    Contains 1 ferric oxidoreductase domain.
  • Post-translational
    modifications

    Glycosylated.
  • Cellular localization

    Membrane.
  • Information by UniProt
  • Database links

  • Alternative names

    • AMCBX2 antibody
    • CGD antibody
    • CGD91-phox antibody
    • CY24B_HUMAN antibody
    • CYBB antibody
    • Cytochrome b 245, beta polypeptide antibody
    • Cytochrome b(558) beta chain antibody
    • Cytochrome b(558) subunit beta antibody
    • Cytochrome b-245 heavy chain antibody
    • Cytochrome b558 subunit beta antibody
    • GP91 PHOX antibody
    • gp91-1 antibody
    • gp91-phox antibody
    • GP91PHOX antibody
    • Heme-binding membrane glycoprotein gp91phox antibody
    • NADPH oxidase 2 antibody
    • Neutrophil cytochrome b 91 kDa polypeptide antibody
    • NOX2 antibody
    • p22 phagocyte B-cytochrome antibody
    • P91 PHOX antibody
    • p91-PHOX antibody
    • Superoxide-generating NADPH oxidase heavy chain subunit antibody
    see all

Images

  • Anti-NOX2/gp91phox antibody (ab80508) at 1 µg/ml + MCF7 cell lysate at 10 µg

    Secondary
    HRP conjugated anti-Rabbit IgG at 1/50000 dilution

    Predicted band size: 65 kDa
    Observed band size: 65 kDa
    Additional bands at: 36 kDa. We are unsure as to the identity of these extra bands.

  • ab80508 staining NOX2/gp91phox in Mouse lung tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 2% milk for 30 minutes at 37°C; antigen retrieval was by heat mediation with a Tris Borate EDTA buffer (pH 8.4). Samples were incubated with primary antibody (1/200 in antibody diluent) for 1 hour at 37°C. A Biotin-conjugated Goat anti-rabbit polyclonal (1/700) was used as the secondary antibody.

    See Abreview

  • ICC/IF image of ab80508 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 (ab80508, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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.

  • ab80508 staining NOX2/gp91phox in Human monocyte-derivive dendritic cells by Flow Cytometry. Cells were fixed with formaldehyde and permeabilized with methanol. The sample was incubated with the primary antibody (1/200) for 30 minutes. An Alexa Fluor®488-conjugated Goat anti-rabbit polyclonal (1/300) was used as the secondary antibody

    See Abreview

  • All lanes : Anti-NOX2/gp91phox antibody (ab80508) at 1/1000 dilution

    All lanes : Mouse liver whole cell lysate

    Lysates/proteins at 100 µg per lane.

    Secondary
    All lanes : HRP-conjugated Goat polyclonal to rabbit IgG at 1/5000 dilution

    Developed using the ECL technique.

    Performed under non-reducing conditions.

    Predicted band size: 65 kDa
    Observed band size: 65 kDa


    Exposure time: 5 seconds


    Primary incubated for 9 hours at 4°C.

    See Abreview

References

This product has been referenced in:

  • Tanaka Y  et al. Adiponectin promotes muscle regeneration through binding to T-cadherin. Sci Rep 9:16 (2019). Read more (PubMed: 30626897) »
  • Hu L  et al. Melatonin decreases M1 polarization via attenuating mitochondrial oxidative damage depending on UCP2 pathway in prorenin-treated microglia. PLoS One 14:e0212138 (2019). Read more (PubMed: 30742657) »
See all 39 Publications for this product

Customer reviews and Q&As

1-10 of 20 Abreviews or Q&A

Question

Dear Technical Support Staff, I am writing regarding a NOX2 antibody (ab80508) which our lab has ordered from your company.  I am currently trying to detect NOX2 expression in respiratory muscles, specifically the diaphragm and sternohyoid in intermittent hypoxia treated rats, by using western blot analysis. It is known that NOX2 is expressed in skeletal muscle under normal conditions, however we have not been able to yield any positive detection of NOX2 expression in sham or IH treated skeletal muscle samples or kidney tissue excised from naive rats or from kidney cell lines which we used as a positive control.  As I am aware you require details of tissue preparation and protocol details for troubleshooting, I will outline the general lab procedure below.   Briefly, the protocol entails culling of the rats after IH or sham exposure. Immediate dissection of the rat and removal of muscles and organs of interest and snap freezing at -80 degrees celsius. Tissues were homogenised and lysed using RIPA buffer. To date I have loaded varying amounts or protein, from 40ug to over 110 ug. I run a 10% gel for 70 minutes at 200v for the electrophoresis stage. I transfer to a nitrocellulose membrane. I have tried both semi dry and wet transfer types. Using Dunn-carbonate for the semi dry and a 15V transfer for the blotting stage for 30mins, and using ice cold Towbin buffer for the wet transfer and a 100V transfer for 1 hour in an ice box. I block in 5% marvel milk TBS-tween (.1% tween) for 1 hour at room temperature. I dilute the primary to 1:1000 in 5% marvel TBS-tween (.1% tween) and incubate overnight at 4 degrees Celsius. I then wash 4 times for 5 minutes in TBS-tween (.1% tween). Then incubation with a secondary anti-rabbit horseradish peroxidase bound antibody (1:2000) for 1 hour at room temperature followed by 4 5minute washes. I then place the membrane in an ECL solution for 5 minutes and expose to photosensitive film and develop.    I am aware that there is a user submitted review on the abcam website for a western blot in skeletal muscle of mice. This was one of the reasons we ordered this antibody from abcam. The protocol seems quite similiar to mine with replacement of mice with rats and also the high protein loading of 200ug, however I still would have expected some sort of a positive result with my current protocol as the antibody is listed to work in rats. I was just wondering if you could offer any suggestions on how to proceed with my experiments as I have invested quite some time into these experiments with little success. What protocol was used to test the antibody? Any suggestions would be greatly appreciated!

Read More
Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. Thank you for providing your protocol information. This information will also allow us to investigate this case internally and initiate additional testing where necessary. After review of that information I can see no apparent reason why this product should not be working in this application. As such, if the product was purchased in the last six months, according to our Abpromise, I would be happy to replace or refund the antibody. In order to do this I do need the original order number or PO number.

Please let me know how you would like to proceed. I look forward to receiving your reply.

Read More
Application
Western blot
Sample
Mouse Tissue lysate - whole (retina)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
retina
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Apr 08 2019

Application
Western blot
Sample
Rat Tissue lysate - whole (muscle)
Gel Running Conditions
Reduced Denaturing (9%)
Loading amount
60 µg
Specification
muscle
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Mr. Baptiste Jude

Verified customer

Submitted Jul 18 2016

Application
Western blot
Sample
Rat Tissue lysate - whole (Skeletal muscle)
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
20 µg
Specification
Skeletal muscle
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 02 2016

Application
Western blot
Sample
Mouse Tissue lysate - whole (Skeletal muscle)
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
20 µg
Specification
Skeletal muscle
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 02 2016

Answer

Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.

I have following further questions for better understanding of the problem;

Lysis buffer
Fresh Protease inhibitors: yes/no
Fresh Phosphatase inhibitors: yes/no
Reducing agent
Boiling for ≥5 min? yes/no
Protein loaded ug/lane or cells/lane
Primary antibody; Concentration or dilution, Incubation time, Incubation temperature
Secondary antibody (Concentration or dilution, Incubation time, Incubation temperature)
Detection method
Type of sample (whole cell lysates, fraction, recombinant protein



Please attach tghe image as well.

Thank you very much for your cooperation. I will look forward to hearing from you soon.

Read More

Answer

While we do not have specific data regarding the possible cross reactivity of this item with other Nox proteins, we are confident of the specificity of this item based on sequence homology of the immunogen against Nox1, 3, 4, 5. Generally we find that a homology of below 85% results in poor reactivity. In this case ab80505 has the following sequence homologies: Nox1: 54.0%, Nox3: 56.0%, Nox4: 43.8%, Nox5: 50.0%

Read More
Application
Western blot
Sample
Mouse Tissue lysate - whole (Skeletal muscle)
Loading amount
50 µg
Specification
Skeletal muscle
Gel Running Conditions
Reduced Denaturing (10% gel)
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Dr. G.K Sakellariou

Verified customer

Submitted Sep 28 2012

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Single isolated FDB skeletal muscle fibres)
Specification
Single isolated FDB skeletal muscle fibres
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton X-100 (for 10 hours)
Blocking step
BSA as blocking agent for 10 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 20°C

Dr. G.K Sakellariou

Verified customer

Submitted Sep 25 2012

Answer

Thank you for contacting us yesterday in regards to the anti-NOX2/gp91phox antibody (https://www.abcam.com/NOX2-gp91phox-antibody-ab80508.html) which is currently unfortunately out of stock.

As discussed over the phone, I have performed a sequence comparison of the immunogen used to raise the alternative antibody https://www.abcam.com/NOX2gp91phox-antibody-ab31092.html, please refer to the attachment of this email (immunogen highlighted in yellow). The holology between the immunogen (a synthetic peptide taken from within residues 100-200 of human NOX2/gp91phox), matchesquite well to the mouse protein.Of the 18 residues used, only 3 do not match. I would therefore expect the antibody to be able to detect the mouse protein. As we have not tested this in house and have no reports of our customers doing so either we cannot guarantee this.

If you would be interested in trying https://www.abcam.com/NOX2gp91phox-antibody-ab31092.htmlfor probing your mouse samples in western blotting I would be able to offer you a testing discount. This would involve you purchasing https://www.abcam.com/NOX2gp91phox-antibody-ab31092.html, testing it, and regardless of whether you find it to work or not, you would be eligible for a free primary antibody of your choice. The only thing we ask in return is for you to let us know the results though submitting an Abreview (this should only take 5-10 minutes). This is a sort of 2 for 1 offer as a reward for providing us with more information about our antibodies.The only constraint is that the antibody to be tested needs to be purchased, tested and the "free" antibody claimed within 4 months. More information on this offer can be found from the following link:

https://www.abcam.com/collaboratordiscount

If you would be interested in this scheme please do let me know as a discount code needs to be generated prior to the purchase of https://www.abcam.com/NOX2gp91phox-antibody-ab31092.html.

Alternatively, we also have a rabbit monoclonal https://www.abcam.com/NOX2gp91phox-antibody-EPR6991-ab129068.html, which hasbeen used to detect the mouse protein with western blotting previously andwould be guaranteed for thisapplication and species under our Abpromise:

https://www.abcam.com/abpromise

I hope this information has been of help.If you require any further information, please do not hesitate to contact us again.

Read More

1-10 of 20 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

Sign up