Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282)

Reviews (1)Q&A (10)References (7)

Overview

  • Product name
    Anti-3-Nitrotyrosine antibody [7A12AF6]
    See all 3-Nitrotyrosine primary antibodies
  • Description
    Mouse monoclonal [7A12AF6] to 3-Nitrotyrosine
  • Host species
    Mouse
  • Specificity
    ab110282 was developed to recognize only protein-bound nitrotyrosine and so is a sensitive tool for measuring protein-specific modifications from oxidative stress.
  • Tested applications
    Suitable for: ICC/IF, Flow Cyt, WB, IP, In-Cell ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Immunogen

    Nitrated KLH (Keyhole Limpet Hemocyanin).

  • Positive control
    • Flow Cyt: HL-60 cells treated with 2 mM peroxynitrite ICC/IF: HeLa cells and Human fibroblast cells treated with 1 mM peroxynitrite WB: nitrated Bovine heart mitochondria; nitrated tyrosine
  • General notes

    This antibody clone is manufactured by Abcam.

    Product was previously marketed under the MitoSciences sub-brand.

    Anti-3-Nitrotyrosine antibody (Alexa Fluor® 488) [7A12AF6] (ab157402)

    Anti-3-Nitrotyrosine antibody (HRP) [7A12AF6] (ab198491)

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab110282 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 a concentration of 1 µg/ml.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

 
WB Use a concentration of 1 µg/ml.
IP Use at an assay dependent concentration.
In-Cell ELISA Use a concentration of 4 µg/ml. (0.4 µg/well).

Target

  • Relevance
    Protein tyrosine nitration results in a post-translational modification that is increasingly receiving attention as an important component of nitric oxide signaling. While multiple nonenzymatic mechanisms are known to be capable of producing nitrated tyrosine residues, most tyrosine nitration events involve catalysis by metalloproteins such as myeloperoxidase, eosinophilperoxidase, myoglobin, the cytochrome P-450s, superoxide dismutase and prostacyclin synthase. Various studies have shown that protein tyrosinenitration is limited to specific proteins and that the process is selective. For example, exposure of human surfactant protein A, SP-A, to oxygen-nitrogen intermediates generated by activated alveolar macrophages resulted in specific nitration of SP-A at tyrosines 164 and 166, while addition of 1.2 mMCO 2 resulted in additional nitration at tyrosine 161. The presence of nitrotyrosine-containing proteins has shown high correlation to disease states such as atherosclerosis, Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis.
  • Alternative names
    • Nitrotyrosine antibody

Images

  • Immunocytochemistry/ Immunofluorescence - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282)
    Immunocytochemistry/ Immunofluorescence - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282)
    Immunocytochemistry image of ab110282 stained Human HeLa cells (A) and fibroblast cells (B, C).
    Cells grown on slides were paraformaldehyde fixed (4%, 20 min) and Triton X-100 permeabilized (0.1%, 15 min). Slides were treated with/without 1 mM peroxynitrite to modify exposed tyrosines to 3-nitrotyrosine. Slides were blocked and incubated with tab110282 at 2 µg/ml overnight at 4°C. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Mouse IgG (H+L) used at a 1/1000 dilution for 1 hour. 10% Goat serum was used as the blocking agent for all blocking steps. For reference, the mitochondria (red) were identified by HSP60 / Alexa Fluor® 594 and DAPI was used to stain the cell nuclei (blue).
    HeLa cells (A) and fibroblast cells (B) show surface modification of tyrosine to 3-nitrotyrosine after exposure to peroxynitrite. While (C) unexposed fibroblast cells show no modification.
  • Flow Cytometry - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282)
    Flow Cytometry - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282)
    HL-60 cells were stained with 1 µg/ml ab110282 following treatment with 2 mM peroxynitrite (blue) or vehicle control (red). No primary antibody control is shown in black. Peroxynitrite modifies tyrosine residues to 3-nitrotyrosine.
  • Western blot - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282)
    Western blot - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282)
    All lanes : Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282) at 1 µg/ml

    Lane 1 : Bovine heart mitochondria
    Lane 2 : Bovine heart mitochondria - nitrated
    Lane 3 : BSA
    Lane 4 : BSA - nitrated
  • Western blot - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282)
    Western blot - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282)
    All lanes : Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282) at 1 µg/ml

    Lane 1 : Bovine heart mitochondria
    Lane 2 : Bovine heart mitochondria - nitrated
    Lane 3 : BSA
    Lane 4 : BSA - nitrated


    Prior to runnning the samples, the membrane was treated with sodium dithionite to reduce nitrotyrosine to aminotyrosine.
  • Western blot - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282)
    Western blot - Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282)
    All lanes : Anti-3-Nitrotyrosine antibody [7A12AF6] (ab110282) at 1 µg/ml

    Lane 1 : Bovine heart mitochondria
    Lane 2 : Bovine heart mitochondria - nitrated
    Lane 3 : BSA
    Lane 4 : BSA - nitrated


    In this experiment, the antibody was first blocked with free nitrotyrosine before being used to blot the membrane.
    The figure shows that ab110282's binding capacity was not inhibited by the free nitrotyrosine, and so only binds to the protein-bound form.

References

This product has been referenced in:
  • Kehm R  et al. Age-related oxidative changes in pancreatic islets are predominantly located in the vascular system. Redox Biol 15:387-393 (2018). Read more (PubMed: 29331666) »
  • Wang X  et al. The TIR/BB-loop mimetic AS-1 prevents non-alcoholic steatohepatitis and hepatic insulin resistance by inhibiting NLRP3-ASC inflammasome activation. Br J Pharmacol 174:1841-1856 (2017). Read more (PubMed: 28306139) »
See all 7 Publications for this product

Publishing research using ab110282? 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 11 Abreviews or Q&A

Western blot abreview for Anti-3-Nitrotyrosine antibody [7A12AF6 ]

 Inconclusive
Abreviews
abreview image
Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (10% gel)
Sample
Guinea pig Tissue lysate - other (cerebellum)
Specification
cerebellum
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Abcam user community

Verified customer

Submitted May 30 2014

Question



Inquiry: Hello, in order to produce a positive control for the Anti-3-Nitrotyrosine-Antibody for Immunfluorescence, we want to treat cells or rather kryosections with peroxynitrite as you advise us to do in your datasheet. Now we would like to inquire how you made up the peroxynitrite working solution (did you solve the peroxynitrite in NaOH?), how did you exactly treat the sections (did you just drop the working solution on the sections?), how long does the peroxynitrite have to incubate and how long is the positive control stable? I hope, you are able to help us. Thank you in advance for your answer. Regards, Kai Zwirnmann

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Answer

Abcam does not offer peroxynitrite. To generate the datasheet data, we purchased peroxynitrite from Millipore as a 110mM solution in 0.3M NaCl and 0.3M NaOH (Millipore cat # 20-107). The Certificate of Analysis for the product has use instructions, e.g. 5mM for 5min, or 1mM from the abcam product figure legend.


We have not specifically tested on IHC, only ICC.

Stability of peroxynitrite is an issue. Once received it should be aliquotted in single use amounts and stored at -80C.

Hope this helps

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Question

Could you send product information/WB protocol sheets for the following Abs of WB:



The PDH cocktail (MSP02/E053),

PDHE1a (Part/LOT#, MSP03/E0668),

mitotyrosine mAB (Part/Lot# MS703/C0578),

MitoProfile Total OXPHOS (CAT/LOT#MS604/D1848/E0944)



We plan to use these Abs for WB of various rat organs/tissues. Any suggestions for pre-caution in using these Abs are appreciated, i.e. Ab dilution, loaded protein amount, ECL system (Amershan vs Pierce etc.).

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Answer

Thank you for contacting us.

Please find the WB protocol used to validate our MitoSciences range of antibodies attached, as well as the pdf datasheets for those products. Keep in mind, we regularly update our online datasheets to reflect the most recent information including additional protocol notes, images, testing new species and applications, and customer feedback.

The recommended concentrations for these antibodies are as follows:

ab110416 (MSP02) MitoProfile Pyruvate dehydrogenase (PDH) WB antibody cocktail: 6 ug/mL
ab110330 (MSP03) anti-Pyruvate Dehydrogenase E1-alpha subunit antibody: 1 ug/mL
ab110282 (MS703) anti-3 Nitrotyrosine antibody: 1 ug/mL
ab110413 (MS604) MitoProfile Total OXPHOS Rodent WB antibody cocktail: 6 ug/mL

While we used Amersham ECL+ for validation of these antibodies, our new Optiblot ECL Max Detect Kit will give a very strong signal with low background, and is suitable for detecting 4.6pg - 4.7ng of protein (ab133408).

I hope this helps, please letme know if you need any additional information or assistance.

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Question

It is not clear to me whether you are sending us another antibody from Abcam to try in our samples.
Also, even if there is no peroxynitration in our samples we should see an absence of signal rather than white bands or dark background.
Could you include an aliquot of a positive control to test with the new antibody?
The secondary antibody we use at the lab is working well with all other primary antibodies, we have tried to detect the Western blots with different lots of secondary antibody and observed the same issues.

Looking forward to finding a solution to this problem,
Thank you,

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Answer

Thank you for your patience.

I am just following up this case. Would you be so kind to confirm how you are getting on?

Currently, we do not have positive control for this antibody. However, I am very pleased to inform you that after discussing this case with some colleagues, the Lab have now generated a nitrated cell lysates and a nitrated BSA as positive control. These will be in the catalogue at the end of May.

Please let me know how you wish to proceed. I look forward to hearing from you soon.

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Question

Hello, here are the answers to your questions:

-I don't think there is a possibility of the gel being overloaded as we used different total protein concentration, as low as 25ug and still the problem persisted. 100ug is tolerated well by the type and thickness of gels we prepare. Also, we checked the transfer with Ponceau Red and it was good in all cases.

-The material used was total lysates from rat cortex tissue, prepared with very standardized protocols we have at the lab, which work well for all other antibodies that we are currently using for Western blotting.

-What we want to see with the antibody is nitrotyrosine in all proteins in general, we will then perform IP to see whether our protein of interest (cytosolic) contains nitrotyrosine residues.

-The samples have not been treated to stimulate nitrosylation because we want to determine whether this occurs in an Alzheimer transgenic model which there is evidence that this is the case. We have also been working with aged animals (14 months old) and there is also evidence that nitrosylation increases with aging (published in rats).

-Were these antibodies tested previously in tissue lysates?

-If you have other anti-nitrotyrosine antibodies could you consider the possibility of sending us a few aliquots to test which one is more suitable to our purposes?

Thank you,

I hope we can find a solution to this problem,

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Answer

Thank you for getting back to me and for answering to my further questions promptly.

I have been discussing this enquiry with my colleague in the Lab and the methods seem to be chosen all look appropriate ECl, milk block etc, Xray film.

It is a bit surprising that there aren’t backgrounds from the rodents immune system (rat circulating IgGs) so you might wish to change the secondary HRP conjugate.With issues like this we are always presuming that there are sufficient nitrated tyrosines in the sample in question to be seen.

Is the level high enough to back up the hypothesis?

This target is a product of the right kind of oxidative stressors and without those there won’t be any modification. To validate this, trying another antibody (rabbit polyclonal) would be a good idea..

To show this antibody works we can use a positive control – add peroxynitrite to the sample – this can be obtained from Millipore. Other donors include SNAP or SIN1 which would create 3-NT.

I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

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Question

We bought 1 Anti-nitrotyrosine Abcam antibody Ab24496 (received 27-10-2011) lot # GR23989-3which didn't work and was replaced by Ab110282 (received 2-12-2011) lot# GR63357-2. In both cases, the detection of specific nitrotyrosine bands was unsuccessful, the blot appeared dark with white bands or just dark with no bands. We have tried the following conditions for both antibodies:

-Different blocking conditions (BSA 5%-10% and milk 5%-10%)
-Protein concentration (25 ug - 100 ug)
-We used rat and mice cortex tissue
-Western blot detected with Regular ECL and ECL plus
-Detection with autoradriographic film and Storm phosphoimager
-Concentration of 1ry antibody 1:100 - 1:5000
-Concentration of 2ry antibody: 1:5000-1:50 000
-Our secondary antibody is working well with other primary antibodies

If we cannot get reimbursed, can we get this antibody instead: Anti-beta Actin antibody - Loading Control (ab8227)

Thank you

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Answer

I am very sorry to hear that both antibodies did not work properly (ab24496 and ab110282).

I can offer you a replacement vial as you wish but it would be important to find out why two antibodies failed to work.

I have read through again the detailed protocols you kindly forwarded to Abcam and I would like to make the following comments/suggestions:

I understand that rat brain lysates were used in these experiments and 25 ug - 100 ug total protein was loaded onto the gel. It may well be tha the gel is overloaded.

Q1: Could you please confirm if total lysates or cellular fractions were used?

Q2: Are you particularly interested in cytosolic protein or any cellular organelle-related proteins?

Q3: Have you treated the samples to stimulate the nitrosylation of teh protein?

As you can see on the Western blot images (ab110282) purified bovine heart mitochondria was used for testing and characterization.

Thank you for your understanding and co-operation in this matter.

I look forward to hearing from you soon.

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Question

We bought 1 Anti-nitrotyrosine Abcam antibody Ab24496 (received 27-10-2011) lot # GR23989-3which didn't work and was replaced by Ab110282 (received 2-12-2011) lot# GR63357-2. In both cases, the detection of specific nitrotyrosine bands was unsuccessful, the blot appeared dark with white bands or just dark with no bands. We have tried the following conditions for both antibodies:

-Different blocking conditions (BSA 5%-10% and milk 5%-10%)
-Protein concentration (25 ug - 100 ug)
-We used rat and mice cortex tissue
-Western blot detected with Regular ECL and ECL plus
-Detection with autoradriographic film and Storm phosphoimager
-Concentration of 1ry antibody 1:100 - 1:5000
-Concentration of 2ry antibody: 1:5000-1:50 000
-Our secondary antibody is working well with other primary antibodies

If we cannot get reimbursed, can we get this antibody instead: Anti-beta Actin antibody - Loading Control (ab8227)

Thank you

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Answer

Thank you for your enquiry. It is very unfortunate that both of these products (ab24496 and ab110282) do not perform as they are expected to do so.

I would like to reassure you that I take your comment seriously. Though, you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.

Could you please provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.

I am particularly interested in the following:

- sample preparation, whole lysate or cellular fraction, buffer used, any stimulation to induce nitro tyrosine etc,

- incubation with the 1ry and the 2ry antibodies i.e. time, temperature,

- blocking time and temperature,

- specification of the secondary antibody (ie. host species, what type of immunoglobulin it was raised against),

- positive control used etc.

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

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Question

Can this antibody be used with rat?

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Answer

Thank you for your enquiry. This antibody is specifically against 3-nitotyorosine only - we are very confident in this. It was generated by nitrated KLH, cross reacts with nitrated BSA (or any other protein) and shows no reaction with un-nitrated KLH. The 3-nitotyrosine is not species specific so this antibody will work with any species, including rat or protein which contains 3 nitrotyrosine. Please rest assured that our Abpromise guarantees that the product will work in species and application as stated on the datasheet.

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Question

Thank you for your help. We would be most grateful if we could try another of your antibodies against nitrotyrosine. We would like to try ab110282 MS703 as it seems to meet more closely our need i.e. it is said that it works in rat homogenates and works for WB as well as other applications. Sincerely,

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Answer

Thank you for your response. This is to let you know that I have placed a new order for you - for one vial of ab110282 as a free of charge replacement (exchange for the original item ab24496) and the new order number is 992370. I hope the second vial will work as it is expected, and please do let me know how you are getting on with this product.  

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Question

ab78406 is unpublished, alternative for rat/IHC-P?

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Answer

Thank you very much for your interest in our anti-Nitrotyrosine antibodies, particularly ab110282. To our knowledge, ab110282 has not been tested in immunohistochemistry on paraffin-embedded sections (IHC-P). Therefore, I can offer a discount off a future purchase if you buy ab110282 now, test it in IHC-P and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free primary antibody. If you are interested in this offer, please follow these steps: 1. Reply to this e-mail to let me know that you would like to proceed and test ab110282 in IHC-P. I will then send a discount code. This code must be issued before purchasing ab so please wait for my reply before ordering. 2. Purchase ab110282 either by phone, fax, or online (www.abcam.com). 3. Test it in IHC-P. 4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out more about our Abreview system, please visit: https://www.abcam.com/abreviews. 5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any primary antibody ordered and the discount code is valid for 4 months after issue. We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab110282 turns out to be unsuitable for IHC-P, you will still receive the discount on your next purchase after your Abreview has been submitted. Please let me know if you have any questions about this offer and I would be happy to help you further. The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.    

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