Overview

  • Product name
    Donkey Anti-Mouse IgG H&L (HRP)
    See all IgG secondary antibodies
  • Host species
    Donkey
  • Target species
    Mouse
  • Tested applications
    Suitable for: Dot blot, ELISA, IHC-P, IHC-Fr, WB, ICC/IFmore details
  • Immunogen

    mouse IgG whole molecule

  • Conjugation
    HRP

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C.
  • Storage buffer
    pH: 7.2
    Preservative: 0.01% Gentamicin sulphate
    Constituents: 0.878% Sodium chloride, 1% BSA, 0.424% Potassium phosphate
  • Concentration information loading...
  • Purity
    Affinity purified
  • Purification notes
    This product was prepared from monospecific antiserum by immunoaffinity chromatography using mouse IgG coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities.
  • Conjugation notes
    Horseradish Peroxidase (HRP)
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab6820 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
Dot blot Use at an assay dependent concentration.
ELISA 1/70000.
IHC-P 1/500 - 1/2000.
IHC-Fr 1/500 - 1/2000.
WB 1/2000 - 1/10000.
ICC/IF 1/500 - 1/2500.

Images

  • Detection of protein levels in LIN-5 mutants.

    (Panel A) Western blots of lysates of yeast clones containing the indicated LIN-5 expression constructs, probed for LIN-5 and tubulin (loading control) levels.

    Protein samples were separated on gradient acrylamide gels and subjected to western blotting on polyvinylidene difluoride membrane. Membranes were blocked with 5% skim milk in PBST for 1 hour at room temperature, or overnight at 4°C for stripped blots. For protein detection, primary antibodies used in this study were: mouse anti–LIN-5 (1:1000) and rabbit anti-Tubulin (1:1000, Abcam) for stripped blots. Secondary antibodies used were: donkey anti-mouse HRP (1:5000, Abcam, ab6820) and goat anti-rabbit HRP (1:5000).

  • IHC image of beta actin staining in human colon formalin fixed paraffin embedded tissue section*.

    The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH 6) for 30 mins. The section was incubated with ab8224, 3 µg/ml overnight at +4°C. An HRP-conjugated secondary (ab6820, 1/2000 dilution) was used for 1 hr at room temperature. The section was counterstained with hematoxylin and mounted with DPX.

    The inset negative control image is secondary-only at 1/500 dilution.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

References

This product has been referenced in:
  • AlHajri SM  et al. Ovine Herpesvirus 2 Glycoproteins B, H, and L Are Sufficient for, and Viral Glycoprotein Ov8 Can Enhance, Cell-Cell Membrane Fusion. J Virol 91:N/A (2017). WB . Read more (PubMed: 28053110) »
  • Fischer MD  et al. Codon-Optimized RPGR Improves Stability and Efficacy of AAV8 Gene Therapy in Two Mouse Models of X-Linked Retinitis Pigmentosa. Mol Ther 25:1854-1865 (2017). Read more (PubMed: 28549772) »
See all 14 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Question

Dear xxxxx,

Thank you for your reply.

I am going to address the issues you have stated in your email one by one:

1. I used Avidin/biotin system not to optimise the experiment, but to show that my methodology for IHC was right. Of course I used different primary antibody with the mentioned system. However, the sample which was used, was from the same person that I used for IHC using Abcam’s secondary antibody (ab6820), which did not work (twice).

2. You have stated that it needs to be established whether COX1 protein is expressed in our hair samples. We already know the gene is expressed in human hair follicle samples from the same source since this has already been shown by RT-PCR and also by qPCR In our laboratory, and now we are aiming to show its protein expression in hair follicles. Therefore we expect to see the expression of COX1 protein. Besides, even if the protein of interest is not expressed in the samples, we always expect to see a background staining(a brownish colour development using AEC kit or green/red background using FITC/TRITC fluorescent antibodies), something which did not occur at all in our experiment which was repeated twice, and this is why we believe your secondary antibody is not working.

3. You have suggested that may be the protein has been harmed during the sample processing or storage. I would like to let you know that the procedure we are using is an established procedure which has been used for years in the University of Bradford and also the sample processing is performed by expert scientists and not junior people. As mentioned in point 1, the prostaglandin F2α receptor was shown by IHC in the sections from the same samples obtained the same time. So I would like to assure you that this not the case.

4. We have purchased these primary antibodies form Abcam:

Mouse monoclonal anti-COX1 (ab22696), mouse monoclonal anti-NAPE-PLD (ab119259), mouse monoclonal anti-FAAH1 (ab54615), rabbit polyclonal anti-FAAH2 (ab103724), and rabbit polyclonal anti-Prostaglandin F2 alpha Receptor (ab126709). As you can see, we need two secondary antibodies: donkey anti-mouse and donkey anti-rabbit. We would really appreciate if you can send two (anti-mouse and anti-rabbit) FITC conjugated secondary antibodies so that I can move my project forward.

Thank you so much for your concern and cooperation. I look forward to receiving your reply and the two above mentioned products.

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Answer

Thank you very much for your reply.

I am sorry that the staining using the Donkey polyclonal Secondary Antibody to Mouse IgG (ab6820) has not performed as expected in your experiment. My aim to gather further information relating to the protocol used and the samples tested has been to try and understand why this is the case and reduce the possible causes. As you have mentioned, the lack of staining could be due to the secondary antibody being inactive but I am sure you can understand it is important to try and understand any other contributory reasons in order to get the experiment working as quickly as possible. I can understand your reluctance at using any further tissue to optimise the antibody further I am therefore able to offer you an alternative free of charge. Would the donkey polyclonal secondary antibody to mouse IgG - H&L (FITC), https://www.abcam.com/Donkey-polyclonal-Secondary-Antibody-to-Mouse-IgG-H-L-FITC-ab97029.html, be suitable?

You mentioned you performed staining with the anti-Prostaglandin F2 alpha Receptor antibody(ab126709) with the same tissue preparations. I am pleased to hear that this staining is performing well. May I ask which secondary antibody you used to perform this staining? Was it the Donkey polyclonal Secondary Antibody to Rabbit IgG (ab6802)?

I look forward to receiving your reply.

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Answer

Thank you for your reply.

I can understand your concern and my aim is to make sure that we resolve this situation as quickly as possible so that you are able to get on with your research. However, in order to achieve this we need to establish why the experiment is not currently working as expected.

Although as you say you have optimised the experiment using the Avadin/biotin system, this is presumably with a different target and different samples which can make a big difference. It therefore needs to be established if the target (COX1) is expressed in your samples and that the protein has not been harmed through the processing or storage of the tissue. You mentioned on the phone that you thought it was a problem of the secondary antibody and this is why i suggested testing this first, before establishing if it was the samples or protocol itself that may need some optimisation in order to achieve the desired results.

I can understand your reluctance to use further tissue samples to perform this and if you would like I can arrange for a new secondary antibody which will work with the ab22696 to be sent to you. However, I must point out that we have not tested the ab22696 in immunofluorescence (ICC/IF) yet and as such would not be able to guarantee its performance. It has currently only been tested in IHC-Fr.

May I just ask though, from your request I understand you are not intending to double stain your samples is this correct? Would you prefer a FITC conjugated secondary antibody?

I look forward to receiving your reply.

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Question

Dear Sir/madam,

Thank you for your email. Please find the answers below:

1- No. We do not fix the samples. After obtaining them from the hospital, they are put immediately in OCT and placed in -80 freezer for future use. This is the way that everybody has been doing for years in the centre of skin sciences at the University of Bradford.

2- The sections were incubated with 3% H2O2 in methanol for 30 min.

3- The AEC kit has been used for a number of other antibodies and it works fine.

4- To see how AEC was used, please see the attached PDF file. The procedure was done exactly according to the protocol provided. The colour development should be seen under the microscope between 10 to 30 min. Three different reagents are provided in the kit and no reagent was made by anybody. To be honest, the protocol is straight forward. Nothing complicated.

5- The primary antibodies which I used to show that my methodology was right were. The optimisation for these two antibodies in order to find the best dilution was previously done by my colleagues. The secondary antibody system was from and Mouse monoclonal anti goat/sheep-biotin. The dilutions used were explained in the questionnaire. To be honest, there was no specific reason that I chose abcam for secondary antibody, and it should not really matter anyway. If Abcam claims that these secondary antibodies are suitable for IHC, then they should really be suitable for IHC.

6- At first, I wanted to do immunofluorescence, and on the data sheet of these two secondary antibodies (ab6802, and ab6820) it is stated that they are suitable for “IF” which stands for immunofluorescence I assume. Then I realised that they are not conjugated with a fluorescein compound. I contacted abcam and talked to one of your colleagues. There I was advised that this was not right and they are not suitable for IF!!!! That was why I wanted to return them. However, I was told that in that case my supervisor would lose some money and that was technically abcam’s problem and not ours. Therefore I decided to go ahead with IHC and keep them. These two antibodies and abcam have put me into too much hassle now.

I hope you have now all the information you may need. Please let me know as quickly as possible what decision you are going to make since my supervisor is waiting for me to get some results.

Kind Regards

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Answer

Thank you for your very detailed reply and sorry for my late reply.

I have been looking into the problems you have encountered and am still investigating the case. I thinkwith the protocol you have been using that there should be staining. However, I think the problem may actually lie with the primary antibody and not the secondary. However, in order to confirm this it would be helpfulif you would be able to try the secondary with another primary antibody which you know to be working as expected. Would this be possible? Or I see you have also purchased the anti-FAAH1 antibody (ab54615), how where you hoping to use this antibody?

I realize this is causing you some inconvenience and once we have identified with which antibody the problem lies I would be happy to replace it.

In order to help you further, I was hoping to try and understand more fully the experiment you are hoping to set up. As you were hoping to perform IF were you aiming to perform double staining with some of your targets? Is this what you are still hoping to do? If so, which targets were you hoping to co-stain?

I understand your confusion over the antibody being listed as being suitable as working in IF. I am looking into where this reference came from and will keep you updated with this information.

I look forward to receiving your reply.

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Answer

Thank you for taking time to completethequestionnaire. The details provided will enableme to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed the information you have provided there are a few additional questions I have if you don't mind. This information will aid in understanding what may be contributing to the problems encountered.

1. Before the skin samples were embedded in OCT were they fixed in anything? For example neutral buffered formalin? Did you perform this step yourself or are you using pre-prepared samples, perhaps the same ones as your colleague prepared?

2. How long were the samples incubated with H2O2 to block the endogenous peroxidases?

3. Have you used the AEC kit with any other antibodies? How has this performed?

4. Could you describe in more detail how the AEC kit was used? How the reagents were prepared and how long the samples were incubated with the reagent? What wash steps did you perform (with which buffersand how many for how long)?

5. I'd be interested to know which antibodies from xxxxx it was that your colleague was using and which secondary antibody? Could you share this information? Why did you choose to move away from the avadin/biotin system?

6. We had an enquiry from you when you purchased these antibodies enquiring if you could send them back to us. Why did you choose to keep them in the end? I believe you wanted to move to using fluorescent detection initially, is this correct?

With this information I should be able to suggest what may be contributing to the problems you have encountered and hopefully suggest a solution.

I look forward to receiving your reply.

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Answer

Thank you for calling us today and reporting the problem you have beenexperiencing with the Anti-COX1 / Cyclooxygenase 1 antibody [CYO-1] (ab22696)used incombination with the Donkey polyclonal Secondary Antibody to Mouse IgG - H&L (HRP) (ab6820). We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

As we discussed on the phone, Ihave attached aquestionnaire so thatI can gather some further information regarding the samples tested and the protocol used. OnceI have receive the completed questionnaire,I will look at the protocol and see if there are any suggestionsI can make that may improve the results.

In particular, could you explain in detail how the samples were collected, fixedand mounted? As well as how the other system used by your colleague was set up (the avadin/biotin system).

I look forward to receiving your reply.

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Question
Answer

Thank you for contacting us.

we tested catalog ab126709in IP, IHC, ICC, and Flow cytometry and it is tested negative. For this reason, we only recommending the product for western blot.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Question
Answer

Thank you for contacting us.

The ab22696 could be used at 1/10 - 1/100 dilution.
ab54615 at 5-10ug/ml
ab103724 at 5-10ug/ml
ab126709 at 1/500-1/1000 dilution.

ab6820 and ab6802 at 1/500-1/2000 dilution.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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