Overview

  • Product name
    Anti-Nitro tyrosine antibody [8C7.3] (Biotin)
    See all Nitro tyrosine primary antibodies
  • Description
    Mouse monoclonal [8C7.3] to Nitro tyrosine (Biotin)
  • Host species
    Mouse
  • Conjugation
    Biotin
  • Tested applications
    Suitable for: IHC-P, WB, ELISA, IP, IHC-Frmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Full length native protein (purified) corresponding to Nitro tyrosine (biotinylated ).

Properties

Applications

Our Abpromise guarantee covers the use of ab24496 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
IHC-P Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml.
ELISA Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.

Target

  • Relevance
    Nitric oxide (NO) is a product of the enzymatic conversion of arginine to citrulline by nitric oxide synthase. NO reacts rapidly with superoxide to form peroxynitrite. At physiological pH and in the presence of transition metals, peroxynitrite undergoes heterolytic cleavage to form hydroxyl anion and nitronium ion, the latter of which nitrates protein tyrosine residues. Thus, the presence of nitrotyrosine on proteins can be used as a marker for peroxynitrite formation in vivo. The presence of nitrotyrosine has been detected in various inflammatory processes including atherosclerotic placques.
  • Alternative names
    • NO tyrosine antibody
    • nTyr antibody

References

This product has been referenced in:
  • Alhalwani AY  et al. Development of a sandwich ELISA with potential for selective quantification of human lactoferrin protein nitrated through disease or environmental exposure. Anal Bioanal Chem 410:1389-1396 (2018). Read more (PubMed: 29214534) »
  • Pais TF  et al. The NAD-dependent deacetylase sirtuin 2 is a suppressor of microglial activation and brain inflammation. EMBO J 32:2603-16 (2013). IHC-P ; Mouse . Read more (PubMed: 24013120) »
See all 2 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

Using a mouse antibody to for mouse IHC can lead to high background if you are detecting the antibody with a secondary anti-mouse IgG antibody, since the anti-IgG may detect endogenous IgG in the sample. You can check for that by incubating a section with just the secondary antibody, which is a recommended negative control for any IHC. There are a few approaches to minimizing the background. One is to directly conjugate the primary antibody (e.g., the anti-nitro tyrosine) to an enzyme (or fluorochrome) so that using an enzyme-conjugated anti-mouse secondary is not necessary.

As it happens, ab24496 is already biotinylated, which was not previously noted on the datasheet. So it could be detected with HRP-conjugated streptavidin, such as ab7403, if you want to visualize the immunostain with light microscopy rather than fluorescent microscopy.

Click here (or use the following: https://www.abcam.com/index.html?datasheet=7403).

We do not have a recommendation for a nitrosylated tyrosine positive control. I suggest looking at the references on the datasheet for ab7408 or the literature in general.

Click here (or use the following: https://www.abcam.com/index.html?datasheet=7408).

This antibody has the most references of the nitro tyrosine antibodies we have and is possibly a better choice than ab24496 but we have not compared them side by side. It is not directly conjugated to anything, so you would need to detect it with a secondary antibody. A good choice for this would be ab98703, which is specific for the isotype subclass of ab7408, IgG2b, and it has been absorbed (purified) to remove reactivity with other subclasses of mouse IgG. Since IgG2b is a relatively rare subclass, background due to staining of endogenous IgG in the tissue section is unlikely to be a problem.

Click here (or use the following: https://www.abcam.com/index.html?datasheet=98703).

Another marker of oxidative stress is 8-hydroxyguanosine. We have several antibodies against this. I suggest ab62623 (which is also mouse IgG2b) or the goat polyclonal antibody ab10802, followed by an anti-goat IgG secondary antibody directly conjugated to HRP and absorbed to remove reactivity with mouse IgG. Please let me know if you need help finding this secondary antibody.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Tissue lysate - whole (Melanoma cancer)
Loading amount
30 µg
Specification
Melanoma cancer
Treatment
5gy Irradiation
Gel Running Conditions
Non-reduced Denaturing (10%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Ms. Seontae Kim

Verified customer

Submitted Jan 02 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Melanoma cancer)
Specification
Melanoma cancer
Fixative
4% Formalin
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 121 Celsius for 10min autoclaved
Permeabilization
Yes - 0.1% SDS
Blocking step
BSA as blocking agent for 15 minute(s) · Concentration: 1% · Temperature: 25°C

Ms. Seontae Kim

Verified customer

Submitted Dec 20 2012

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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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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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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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

We are trying to do western blots, a technique commonly used in our lab, using your antibody against anti-nitrotyrosine (ab24496) and its not working. Our samples are homogenates of rat brain, prepared as usual in cell lysis buffer (from cell signaling) supplemented with complete protease inhibitor cocktail (from Roche). We are running in denaturing conditions (with SDS) and we tried to transfer to PVDF and nitrocellulose. According to Rouge Ponceau staining of the membranes, our proteins are fine. We tried blocking in different conditions: non-fat milk 5-10% or BSA 5% (from 2 to 16 hours) We tried different concentrations of this primary (ab24496): 1/250 to 1/5000. We tried different concentrations of the secondary (peroxidase goat anti-mouse): 1/5000 to 1/50000. This secondary antibody works beautifully with other primary antibodies. The best signal we can get is white bands on dark background (see attached document). Can you please tell us the conditions in which this antibody should work. I didnt find any published papers using this antibody. Thanks for a fast reply! Abcam WB questionnaire 1) Abcam product code: ab24496 (mouse monoclonal antibody anti-nitrotyrosine) 2) Abcam order reference number or product batch number: lot:GR23989-3 3) Description of the problem : Unable to obtain a good signal despite trying several different conditions. The best signal we can get is white bands on dark background (see figure) 4) Sample preparation: Type of sample (whole cell lysates, fraction, recombinant protein…): total rat brain homogenates Lysis buffer : cell lysis buffer 1X (from cell signaling) Protease inhibitors: complete protease inhibitor cocktail (from Roche) Phosphatase inhibitors: No Reducing agent : SDS 2% and b-mercaptoethanol 1% Boiling for ≥5 min? yes Protein loaded ug/lane or cells/lane : tried from 25-100 ug (most often 70 ug) Positive control :yes (brain extracts of transgenics rats were high levels were demonstrated previously by our team, using the same antibody (Bruno et al Neurobiol aging 2011 (rats); Bruno et al J Neuropathol Exp Neurol 2009 (human)) Negative control :no 5) Percentage of gel: 12% Type of membrane : tried PVDF or nitrocellulose Protein transfer verified :yes with rouge ponceau Blocking agent and concentration: tried non-fat milk 5-10% or BSA 5% in TBS-T 0.1% Blocking time: tried from 2 to 16 hours Blocking temperature : if 2-5 hours: room temperature; if 16 hours: 4 degrees 6) Primary antibody (If more than one was used, describe in “additional notes”) : Concentration or dilution: tried 1/250 to 1/5000 Diluent buffer : tried non-fat milk 5-10% or BSA 5% in TBS-T 0.1% Incubation time: 2h at room temperature or 16h at 4degrees Incubation temperature: 2h at room temperature or 16h at 4degrees 7) Secondary antibody: peroxidase goat anti-mouse) Species: goat Reacts against: mouse Concentration or dilution : tried 1/5000 to 1/50000 Diluent buffer :TBS-T 0.1% Incubation time: 1 hour Incubation temperature: room temperature Fluorochrome or enzyme conjugate: peroxidase (HRP) 8) Washing after primary and secondary antibodies: Buffer: TBS-T 0.1% Number of washes: after primary, 5 washes in 1 hour, after secondary, 3 washes in 30 min Detection system: ECL plus on autoradiographic film or STORM Do you obtain the same results every time? Either no signal or white bands on dark backgroung What steps have you altered to try and optimize the use of this antibody? All except homogenate preparation Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem

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Answer

Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this product. Looking at the WB image you have kindly attached, it is very likely that ab24496 is inactive since you have nice signal with an alternative antibody after reprobing using the same samples. I would like to reassure you that our Abpromise applies to your complaint since you purchased this product within the guarantee period. This means that in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased. I could offer you either a new vial as a free of charge replacement or a credit note which you can use in the future. Please do let me know how you wish to proceed with this enquiry. I look forward to hearing from you and hope to solve this problem as soon as possible.  

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