Biotin Anti-Nitrotyrosine 抗体 [8C7.3] (ab24496)

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/Streptavidin-HRP-ab7403.html).

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/ab7408.html).

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/Goat-Mouse-IgG2b-heavy-chain-HRP-preadsorbed-ab98703.html).

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.

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.

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.

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.

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.  

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.