Overview

  • Product name
    HRP Conjugation Kit
  • Product overview

    HRP Conjugation Kit ab102890 uses a simple and quick process to conjugate an antibody to HRP. It can also be used to conjugate other proteins or peptides.


    To conjugate an antibody to HRP using this kit:
    - add modifier to antibody and incubate for 3 hrs
    - add quencher and incubate for 30 mins
    The conjugated antibody can be used immediately in WB, ELISA, IHC etc. No further purification is required and 100% of the antibody is recovered for use.


    Learn about buffer compatibility below; for incompatible buffers and low antibody concentrations, use our rapid antibody purification and concentration kits. Use the FAQ to learn more about the technology, or about conjugating other proteins and peptides to HRP.

  • Notes

    Amount and volume of antibody for conjugation to HRP

     Kit size   Recommended
    amount of antibody
    1 
    Maximum
    amount of antibody
    Maximum antibody
    volume2
    30 µg   3 x 10 µg  3 x 40 µg 3 x 10 µL
    100 µg   100 µg   400 µg 100 µL
    300 µg   3 x 100 µg  3 x 400 µg 3 x 100 µL
    1 mg   1 mg 4 mg 1 mL

    1 Recommended amount of antibody will give an HRP:antibody molar ratio of 4:1. Antibodies often also perform well at the maximum amount of antibody which is 1:1 molar ratio.

    2 Ideal antibody concentration is 1mg/ml. 0.5 - 1 mg/ml can be used if the maximum antibody volume is not exceeded. Antibodies > 5mg/ml or < 0.5 mg/ml should be diluted /concentrated.

     

    Buffer Requirements for Conjugation

    Buffer should be pH 6.5-8.5.

    Compatible buffer constituents
    If a concentration is shown, then the constituent should be no more than the concentration shown. If several constituents are close to the limit of acceptable concentration, then this can inhibit conjugation.

    50mM / 0.6% Tris1 0.1%/1% BSA2 50% glycerol
    0.1% sodium azide3 PBS Potassium phosphate
    Sodium chloride HEPES Sucrose
    Sodium citrate EDTA Trehalose

    1 Tris buffered saline is almost always ≤ 50 mM / 0.6%
    2 1% BSA gives lower quality conjugates, BSA can also interfere with the use of the conjugated antibody in tissue staining.
    3 Sodium azide irreversibly inhibits HRP; antibodies with azide should be purified before using the HRP conjugation kit.

    Incompatible buffer constituents

    Thiomerosal Proclin Glycine
    Arginine Glutathione DTT

    If a constituent of the buffer containing your antibody or protein is not listed above, please check the FAQ or contact us.

    Only purified antibodies are suitable for use, ie. where other proteins, peptides, or amino acids are not present: antibodies in ascites fluid, serum or hybridoma culture media are incompatible.

  • Tested applications
    Suitable for: Conjugationmore details

Properties

Applications

Our Abpromise guarantee covers the use of ab102890 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
Conjugation Use at an assay dependent dilution.

Images

Protocols

References

This product has been referenced in:
  • Park JK  et al. Evaluation of Preexisting Anti-Hemagglutinin Stalk Antibody as a Correlate of Protection in a Healthy Volunteer Challenge with Influenza A/H1N1pdm Virus. MBio 9:N/A (2018). Read more (PubMed: 29362240) »
  • Tan X  et al. Rapid Mouse Follicle Stimulating Hormone Quantification and Estrus Cycle Analysis Using an Automated Microfluidic Chemiluminescent ELISA System. ACS Sens 3:2327-2334 (2018). Read more (PubMed: 30335974) »
See all 6 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Answer

Thank you for contacting us.
As my colleague mentioned, we do not currently carry any antibodies to the Aftose virus. You may be able to find one by searching biocompare.com.
For the other products you mentioned, we do have a number of HRP conjugated secondary antibodies, as well as conjugation kits that can be used to label primary antibodies with HRP:
www.abcam.com/EasyLink
For an HRP substrate, we offer DAB detection kits for colorimetric studies (such as ab94665), and ECL substrates for western blotting (such as ab65623).
I hope this helps, please let me know if you need any additional information or assistance.

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Answer

Thank you for your inquiry. In case the customer would like to perform a sandwich ELISA (sELISA) for the EBV viral capsid antigen, I would suggest to test the antibody clones [2E08] and [8.F.90] (ab48414 and ab32803, see below). To our knowledge, this pair has not yet been tested in sELISA, and therefore, I can offer a discount off a future purchase if the customer buys them now, test them in sELISA 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 the feedback. The discount would be to the value of 1 free primary antibody each. The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount. Please let me know if the customer is interested in this offer and I would be happy to help you further. If these antibodies are used in sELISA, one of them (the detection antibody) needs to be conjugated to an enzyme such as HRP and we would therefore recommend our EasyLink conjugation kits ab102889 or ab102890 (www.abcam.com/EasyLink or see below). If the customer is interested in one of these, I would be pleased to offer a discount. Unfortunately, we do not have any other antibodies available against EBV Ea-R p17 than ab64281 and against EBV Ribonucleotide Reductase than ab6504, respectively. Therefore, I cannot give a recommendation for a suitable paired antibody to use in sELISA. Having said this, I could recommend the HRP-conjugated secondary antibodies ab98693 (for the mouse-IgG1 antibody ab6504) and ab98698 (for the mouse-IgG2a antibody ab64281) in case the customer would like to set up a conventional indirect ELISA (see below). Using a secondary antibody amplifies the signal and this might be useful for proteins of low abundance. https://www.abcam.com/index.html?datasheet=32803 https://www.abcam.com/index.html?datasheet=32803. https://www.abcam.com/index.html?datasheet=102889 https://www.abcam.com/index.html?datasheet=102889. https://www.abcam.com/index.html?datasheet=102890 https://www.abcam.com/index.html?datasheet=102890. https://www.abcam.com/index.html?datasheet=98693 https://www.abcam.com/index.html?datasheet=98693. https://www.abcam.com/index.html?datasheet=98698 https://www.abcam.com/index.html?datasheet=98698. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question

LOT NUMBER GR29857 ORDER NUMBER 941272 DESCRIPTION OF THE PROBLEM No bands detectable in western blot (repeated twice), also after incubation with 2° antibody. After incubation with the first wash fraction from purification (instead of the HRP-conjugate) and the 2° antibody, there was a weak signal. SAMPLE Protein extract from rat intestinal mucosa (separate extracts for duodenum, jejunum, ileum, caecum, colon) PRIMARY ANTIBODY Anti-Vitamin D Receptor antibody [9A7gammaE10.4] (ab54387) I expected the 100 ug of antibody to be in the HRP-conjugate, which was 480 ul. According to this, I used a dilution of 1:100, incubation in 1% BSA overnight at 4°C, Three wash steps with PBST Loading control: mouse anti GAPDH (HRP), ab9482, Lot GR44318 POSITIVE AND NEGATIVE CONTROLS USED None ANTIBODY STORAGE CONDITIONS Storage at 4 °C on arrival, antibody purification (with the purification kit, ab102783, Lot GR36646) according to protocol (followed it exactly) one day after arrival and conjugation with HRP (EasyLink conjugation kit, ab102890, Lot GR51319) according to the protocol the following day, since then storage at 4°C. SAMPLE PREPARATION RIPA buffer with PMSF, leupeptin and aprotinin for extraction, incubation of samples with loading buffer for 5 minutes at 95°C before loading. AMOUNT OF PROTEIN LOADED 40ug/well ELECTROPHORESIS/GEL CONDITIONS Reducing 12% gel, 120 V TRANSFER AND BLOCKING CONDITIONS Transfer: wet, 135 mA, 90 minutes, Tris-glycine with 20% methanol, nitrocellulose membrane Ponceau S Red for protein detection (very nice signal) Blocking: 5% BSA in 0.1 % PBS Tween for 60 minutes at room temperature SECONDARY ANTIBODY GE healthcare amersham bisocience, goat anti-rat IgG, HRP-linked Dilution 1:5000 in 1% BSA, incubation for 90 minutes at room temperature HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No WHAT STEPS HAVE YOU ALTERED? the third time, I used the wash fraction from purification instead of the assumed HRP-antibody-conjugate, which gave me a weak signal. ADDITIONAL NOTES The loading control worked fine. i attached an image of the first attempt (loading control + membrane after incubation with HRP-antibody-conjugate) and of the third (membrane after incubation with wash fraction)

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Answer

Thank you for contacting us. I am sorry to hear you are experiencing difficulties with some of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. In this case the first thing I investigated was whether the purification kit is compatible with rat IgG, especially since a weak signal was observed with the wash fraction. In this case ab102783 contains protein A resin which shows no affinity for rat IgG therefore is not being effectively purified. This explains why there is a weak signal from the wash fraction as the antibody is being eluted and not captured effectively. Reviewing recent correspondence I can see that you had been in touch with us previously and directed to the purification and conjugation kits. I would like apologize that these products are not working for you and for any inconvenience that may have caused. I would be happy to reimburse you if we misled you in any way. Cross-reactivity with the rat samples was something you were concerned about as ab54387 is a rat antibody, however I do not think this would be a major problem. This may be true in IHC but in WB it is unlikely that endogenous Ig's will be present. Have you tried using ab54387 and a suitable secondary to check for cross-reactivity in the sample? If this is a problem there are some alternative primary antibodies I would like to recommend: ab3508 Anti-Vitamin D Receptor antibody https://www.abcam.com/index.html?datasheet=3508 ab80666 Anti-Vitamin D Receptor antibody [9A7 gamma .E10.E4] - BSA and Azide free https://www.abcam.com/index.html?datasheet=80666 ab109234 Anti-Vitamin D Receptor antibody [EPR4552] https://www.abcam.com/index.html?datasheet=109234 ab80666 although again this is a rat monoclonal, it is BSA and Azide free therefore the purification kit would not be necessary and the antibody could be conjugated directly with the EasyLink HRP conjugation kit. This is something you may be interested in to remove the need for a compatible secondary antibody. Again I am sorry for any inconvenience and look forward to receiving your reply, so that we can resolve this matter as soon as possible.

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