Overview

  • Product namePE/Cy5® Conjugation Kit
  • Product overview

    Abcam's PE/Cy5 Conjugation kit provides a simple and quick process to conjugate your primary antibodies with PE/Cy5. PE/Cy5 is a tandem conjugate: PE (energy donor) has an excitation wavelength of 565nm and Cy5 (energy acceptor) has an emission wavelength of 666nm.

    The conjugated antibody can be used straight away in WB, ELISA, IHC etc

    Learn more about buffer compatibility, protein/secondary antibody conjugation and labelling chemistry in our FAQs.

     

  • Notes

    Amount and volume of antibody

    In view of the large size of the PE/Cy5 label, the amount of antibody used in a labeling reaction with the tandem must always be less than the amount of PE/Cy5, in order that the PE/Cy5 is in a slight molar excess. The best ratio for any new antibody reagent must be determined by experimentation but 50-60µg of IgG antibody for every 100µg of PE/Cy5 usually gives optimal results. 50µg of antibody corresponds to an Antibody:PE/Cy5 molar ratio of 1:1.

    The volume in which the antibody is added ideally should be around 400µl. Where the concentration of antibody is relatively low, and where it is impractical to concentrate the antibody, up to twice the volume stated above may be added without any significant loss in conjugation efficiency.

     

    Protocol

    • Before you add antibody to the PE/Cy5 mix, add 100µl of Modifier reagent for 1ml of antibody to be labeled. Mix gently.
    • Remove the screw cap from the vial of PE/Cy5 mix and pipette the antibody sample (with added Modifier) directly onto the lyophilised material. Resuspend gently by withdrawing and re-dispensing the liquid once or twice using a pipette.

     

    • Place the cap back on the vial and leave the vial standing for 3 hours in the dark at room temperature (20-25°C). Alternatively, and often more conveniently, conjugations can be set up and left overnight, as the longer incubation time has no negative effect on the conjugate.

     

    • After incubating for 3 hours (or more), add 1µl of Quencher reagent for every 10µl of antibody used. The conjugate can be used after 30 minutes.

     

    Storage of conjugates

    For any new conjugate, initial storage at 4°C is recommended. A preservative may be desirable for long-term storage. Other storage conditions (e.g. frozen at -70°C or stored at -20°C with 50% glycerol) may also be satisfactory. The best conditions for any particular conjugate must be determined by experimentation.

    Sample Buffer

    Ideally, the antibody to be labeled should be in 10-50mM amine-free buffer pH range 6.5 to 8.5. However, many buffers outside these limits of concentration and pH can be accommodated. Modest concentrations of Tris buffer are also tolerated.

    Amine-free buffers, including MES, MOPS, HEPES and phosphate are compatible if they are in the concentration range 10-50mM and have pH values in the range 6.5-8.5, as the addition of Modifier provides the conditions necessary for efficient conjugation. Common non-buffering salts (e.g. sodium chloride), chelating agents (e.g. EDTA), and sugars may be present, as they have no effect on conjugation efficiency. Azide (0.02-0.1%) has little or no effect. If the amine-free buffer is relatively concentrated and outside the pH range 6.6-8.5 you may need to add more Modifier for each 10µl of antibody. Excess Modifier is provided so that you can check the pH of the buffer after the addition of the modifier. Ideally the pH should be around 7.3-7.6, though efficient conjugation occurs anywhere between pH 6.8 and 7.8. Avoid buffer components that are nucleophilic, as these may react with the kit chemicals. If your buffer contains primary amines (e.g. amino acids, ethanolamine) and/or thiols (e.g. mercaptoethanol, DTT), you should consider using our Concentration and Purification Kits (ab102776 or ab102783). (Note: Unusually, for an amine, Tris has little effect on conjugation efficiency as long as the concentration is 20mM or less).

    Disclaimer

    Labeling of the antibody will not work if the conjugation blocks the active paratope. This situation is rare.

     

    This material is also subject to proprietary rights of

  • Tested applicationsConjugation more details

Properties

  • Alternative names
    • Phycoerythrin

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 Notes
Conjugation Use at an assay dependent dilution.

PE/Cy5® Conjugation Kit images

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

Protocols

References for PE/Cy5® Conjugation Kit (ab102893)

ab102893 has not yet been referenced specifically in any publications.

Product Wall

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: http://www.abcam.com/antibody-purificat...

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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 ha...

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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, prima...

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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

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.

Use ou...

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"