Overview

  • Product name
  • Description
    Rabbit polyclonal to NOX1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, WB, ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Human, Pig
  • Immunogen

    Synthetic peptide corresponding to C terminal residues of human NOX1.

Properties

Applications

Our Abpromise guarantee covers the use of ab55831 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.
WB Use at an assay dependent concentration. Predicted molecular weight: 65 kDa.Can be blocked with NOX1 peptide (ab194192).
ELISA Use at an assay dependent dilution.

Target

  • Function
    NOH-1S is a voltage-gated proton channel that mediates the H(+) currents of resting phagocytes and other tissues. It participates in the regulation of cellular pH and is blocked by zinc. NOH-1L is a pyridine nucleotide-dependent oxidoreductase that generates superoxide and might conduct H(+) ions as part of its electron transport mechanism, whereas NOH-1S does not contain an electron transport chain.
  • Tissue specificity
    NOH-1L is detected in colon, uterus, prostate, and colon carcinoma, but not in peripheral blood leukocytes. NOH-1S is detected only in colon and colon carcinoma cells.
  • Sequence similarities
    Contains 1 FAD-binding FR-type domain.
    Contains 1 ferric oxidoreductase domain.
  • Cellular localization
    Cell projection > invadopodium membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • GP91 2 antibody
    • Mitogenic oxidase (pyridine nucleotide dependent superoxide generating) antibody
    • Mitogenic oxidase 1 antibody
    • MOX-1 antibody
    • MOX1 antibody
    • NADH/NADPH mitogenic oxidase subunit P65 MOX antibody
    • NADH/NADPH mitogenic oxidase subunit P65-MOX antibody
    • NADPH oxidase 1 antibody
    • NADPH oxidase 1 variant NOH 1L antibody
    • NADPH oxidase homolog 1 antibody
    • NOH 1 antibody
    • NOH-1 antibody
    • NOH1 antibody
    • NOX-1 antibody
    • Nox1 antibody
    • NOX1_HUMAN antibody
    • RP1 146H21.1 antibody
    see all

Images

  • ICC/IF image of ab55831 stained HepG2 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 (ab55831, 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.

References

This product has been referenced in:
See all 20 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Application
Immunoprecipitation
Immuno-precipitation step
Other - Gamma-bind plus protein A/G (half/half)
Sample
Human Cell lysate - whole cell (KERATINOCYTES, Hacat B)
Specification
KERATINOCYTES, Hacat B
Treatment
inhibitor of nox 50´M
Total protein in input
1000 µg

Dr. houssam raad

Verified customer

Submitted Mar 12 2015

Abreviews
Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Non-reduced Denaturing (Any Kd)
Sample
Rat Tissue lysate - whole (brain)
Specification
brain
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Jun 13 2014

Application
Western blot
Loading amount
40 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Tissue lysate - whole (Liver)
Specification
Liver
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jun 06 2014

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
Sample
Human Cell (colon cancer cell)
Specification
colon cancer cell
Permeabilization
Yes - 0.1% Tween 20
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Feb 28 2014

Answer

I am very pleased to hear you would like to accept our offer and test ab55831 in mouse. This code will give you $347 off your next order before the expiration date. To redeem this offer, please submit an Abreview for mouse and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.

For more information on how to submit an Abreview, please visit the site: https://www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: https://www.abcam.com/abtrial.

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Answer

Thank you for your phone call.
DISCOUNT CODE: xxx for ab65813
DISCOUNT CODE: xxx for ab31092
DISCOUNT CODE: xxx for ab55831
Expiration date: xxx
I am very pleased to hear you would like to accept our offer and test these 3 antibodies in flow cytometry and/or mouse. Each code will give you: 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for flow cytometry and/or mouse and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.
Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.
Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.
The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Question

This customer has purchased ab55831 (Anti-NOX1 antibody) and has conducted the wb several times with mouse sample. The results show multiple bands, therefore, he wants to ask for your help to modify her experiment step, could you please offer any suggestion to help her?

I also attached her image in this letter and her experiment step as follow:



1. Order details:


Batch number: 11111111

Po: aaaaaaaa

Abcam product code: ab55831

Antibody storage conditions (temperature/reconstitution etc): -20




2. Please describe the problem (high background, wrong band size, more bands, no band etc).

More bands

3. On what material are you testing the antibody in WB?

· Species: mouse

· What’s cell line or tissue :aorta

· Cell extract or Nuclear extract: cell extract

· Purified protein or Recombinant protein:



3. The lysate


How much protein was loaded: 30micro gram

What lysis buffer was used: np40



What protease inhibitors were used: Roche cocktail

What loading buffer was used: 6x sample dye

Phosphatase inhibitors Na3VO4

Did you heat the samples: temperature and time: 95/5 min




4. Electrophoresis/Gel conditions/ Transfer conditions


Reducing or non reducing gel: non

Reducing agent:

Gel percentage : 10and 15

Transfer conditions: (Type of membrane, Protein transfer verified): 100v 1hr




5. Blocking conditions


Buffer : TBS

Blocking agent: 5%milk:

Incubation time: 1hr

Incubation temperature: RT




6. Primary Antibody


Species: R

Reacts against:


· At what dilution(s) have you tested this antibody: 1:500 or 1:1000

· What dilution buffer was used: 5% milk /TBS

· Incubation time: 4℃ on

· Incubation temperature:

· What washing steps were done:



7. Secondary Antibody


Species:

Reacts against: Rabbit

At what dilution(s) have you tested this antibody: 1:5000

Incubation time: 1hr

Wash steps: 10min with 1% TBST for 3 times

Fluorochrome or enzyme conjugate:

Do you know whether the problems you are experiencing come from the secondary?




8. Detection method
ECl,



9. Did you apply positive and negative controls along with the samples? Please specify. No



10. Optimization attempts

· How many times have you tried the Western? 2 times

· Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): yes

· Do you obtain the same results every time e.g. are background bands always in the same place? yes

· What steps have you altered? Prolong the wash step



Could you please help this customer to solve the problem?

Thanks for your kindly help

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Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab55831 Anti-NOX1 antibody.

As this is a multipass membrane protein, which tent to aggregate in the loading buffer when boiled at high temperature due to their hydrophobic nature, I would like to recommend lowering the boiling temperature down to 60C and prolonging the boiling time up to 10 min. In addition, use DTT instead of beta-mercaptoethanol as this kind of aggregation is favored by b- ME.
Most of our antibodies are tested and optimized to work under reducing conditions, and we usually state on our datasheet if the gel should be run under non-reducing conditions. I would therefore suggest to run a SDS-PAGE under reducing conditions. Otherwise it may be that the antibody may not be able to detect its target and binds non-specific.
This protein has two glycosylation sites, that means that bands can occur different from the expected molecular weight. In addition,all three isoforms of this protein share the C-terminus, so it is possible that the antibody detects those two additional variationsas well. Isoform NOH-1LVis also glycosylated.
In order to facilitate the weight of the different bands, I would suggest to decrease the gel percentage down to 8%.
In order to rule out a cross-reactivity of your secondary antibody, i would suggest running a non-primary control.
Addition of detergents to diluted antibodies can significantly reduce background on the blot. I would suggest to incubate the antibody in a 1:1000 dilution in 5% milk TBST solution. 1% Tween is very high for the wash solution and incubation, therefore, I would suggest to lower it down to 0.1%. Avoidexposing the membrane to detergent until blocking is complete, as this may cause high membrane background.



Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

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Answer

Thank you for contacting Abcam regarding ab55831. I am sorry that you are experiencing difficulties with this antibody in WB. I have reviewed the protocol information you provided and I would like to make some suggestions to help improve your results with this product. Additionally, I have a few questions to help clarify how you are using the antibody. Regarding the sample - what type of sample are you using? What is the species and tissue/cell type? The antibody has only been tested in human and mouse, so it is important that you are using one of these. Regarding the gel conditions - why are you running this under non-reducing conditions? Does your loading buffer contain any DTT or b-mercaptoethanol? Our testing conditions indicate that the western blot be run under reduced, denatured conditions. Additionally, I would recommend increasing the amount of protein loaded to 60 - 100ug. What type of transfer are you performing (wet or semi-dry)? Have you confirmed the efficiency of your transfer to the membrane by Ponceau S staining? If you have not done so already, I would also recommend increasing the concentration of primary antibody and confirming that your secondary antibody is working with other primary antibodies. I hope this information is helpful. Please do not hesitate to contact us if you have any additional quesitons or if these suggestions do not improve your results with this antibody.

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Question
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 (different lot number of ab55831) with the order number ******. 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.

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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. I appreciate the time you have spent on these experiments and would be pleased to arrange a replacement of ab55831 with a different lot number or with one unit of the alternative anti-NOX1 ab78016. If you prefer, I can also issue a refund or a credit note in compensation. I look forward to hearing from you with details of how you would like to proceed.  

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1-10 of 12 Abreviews or Q&A

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