Overview

  • Product name
    PE/Cy5® Conjugation Kit
  • Product overview

    PE/Cy5® Conjugation Kit ab102871 uses a simple and quick process to conjugate an antibody to PE/Cy5®. It can also be used to conjugate other proteins or peptides.


    To conjugate an antibody to PE/Cy5® 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 PE/Cy5®.


     

  • Notes

    Amount and volume of antibody for conjugation to PE/Cy5®.

     Kit size   Recommended 
    amount of antibody
    1 
    Maximum antibody 
    volume2
    30 µg   3 x 10 µg 3 x 10 µL
    60 µg   60 µg  60 µL
    180 µg   3 x 60 µg 3 x 60 µL
    600 µg   600 µg 600 µL

    Kit is designed to give a 1:1 molar ratio of antibody to PE after conjugation.

    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.

    The selling size of this product has been changed – it is now based on the amount of antibody that can be conjugated with the kit, not the amount of PE mix provided. The amount of antibody advised that can be used with the kit has also been updated to reflect what will give the best conjugation results. The quantity and formulation of reagents provided have not changed, if you have been previously using the kit successfully with a different amount of antibody, there is no need to change the way that you are using the kit.

     

    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 azide 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.

    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 ab102893 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

  • This illustration demonstrates a general procedure and will slightly vary dependent on the conjugate used.

Protocols

References

This product has been referenced in:
  • Nielsen CM  et al. Impaired NK Cell Responses to Pertussis and H1N1 Influenza Vaccine Antigens in Human Cytomegalovirus-Infected Individuals. J Immunol 194:4657-67 (2015). Read more (PubMed: 25855356) »
  • White MJ  et al. Differential activation of CD57-defined natural killer cell subsets during recall responses to vaccine antigens. Immunology 142:140-50 (2014). Read more (PubMed: 24843874) »
See all 2 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Answer

We wouldn’t recommend using the kits with an antibody at such a low concentration as the conjugation reaction will result in an over labelled antibody. We offer antibody concentration and purification kits: https://www.abcam.com/antibody-purification-kit-protein-a-ab102784.html or

https://www.abcam.com/antibody-purification-kit-protein-g-ab128747.html

Please select the kit based on the species of the secondary antibodies. Please see the table at the bottom of this webpage https://www.abcam.com/index.html?pageconfig=resource&rid=11385

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Question
Answer

One more thing I learned from the lab, if you are still concerned about the effect of the EasyLink reagents on live cells:

"We commonly perform overnight conjugations here without adding the Quencher in the morning. The reactive chemicals have a short half-life, and should already be inactivated after an overnight conjugation. Unfortunately, we do not have any data on the effect of the kit components on live cells."

As I mentioned in my earlier e-mail, many customers follow the shorter incubation protocol on the datasheet and stain live cells successfully.

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Question
Answer

Following up my previous message, the laboratory does not have toxicity data but the assumption is that the reagents are not harmful to live cells, given that many customers have reported using Easy-Linked antibodies with their live cell samples, primarily in flow cytometry. Please let me know if you have any questions or concerns.

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Answer

Questions of this kind are best directed towards our customer service team, not to us in scientific support. Please send your enquiry to sales@abcam.com

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Answer

Thank you for contacting us.

Right now, we have two kits in stock; the lot. number is 1226982.

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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