Overview

  • Product name
    PerCP/Cy5.5® Conjugation Kit
  • Product overview

    PerCP/Cy5.5® Conjugation Kit ab102911 uses a simple and quick process to conjugate an antibody to PerCP/Cy5.5®. It can also be used to conjugate other proteins or peptides.


    To conjugate an antibody to PerCP/Cy5.5® 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 PerCP/Cy5.5®.


     

  • Notes

    Amount and volume of antibody for conjugation to PerCP/Cy5.5®

     Kit size   Recommended maximum
    amount of antibody
     
    Maximum antibody 
    volume1
    30 µg   3 x 10 µg 3 x 10 µL
    100 µg   100 µg  100 µL
    300 µg   3 x 100 µg  3 x 100 µL
    1 mg   1 mg 1 mL

    1 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 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 

  • Tested applications
    Suitable for: Conjugationmore details

Properties

Applications

Our Abpromise guarantee covers the use of ab102911 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:
  • Mutoji K  et al. TSPAN8 Expression Distinguishes Spermatogonial Stem Cells in the Prepubertal Mouse Testis. Biol Reprod 95:117 (2016). Conjugation . Read more (PubMed: 27733379) »
See 1 Publication for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Answer

The conjugation is effective within the range 1-4 mg/ml, and less effective ouside of it. So, you still may be able to obtain some signal with a conjugation of a 0.5mg/ml antibody but it will be relatively weak. We recommend concentrating the antibody, for instance with these filter units, ab102778:

https://www.abcam.com/index.html?datasheet=102778

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Question
Answer

ab72479:
Like most phycobiliprotein conjugates, the ratio of the fluor to the antibody (F/P) can vary slightly from lot to lot due to the nature of the conjugation chemistry and the methods used to characterize the final product, but it is typically around 1:1. The F/P for the latest batch of the D5-1722 mouse anti-HA-RPE is 0.83 PE/Ab, which essentially falls into that 1:1 zone.

ab102911:
The PerCP/Cy5.5 kit should result in an antibody:label ratio similar to that following labeling with a PerCP kit. The advice we normally give is as follows:
The amount of antibody used for labeling should be the same as the pack size of LL-PerCP-Cy5.5 e.g. For 100mg LL-PerCP-Cy5.5 add 100mg of antibody. The volume in which the antibody is added ideally should be 4ml-10ml (10mg pack size, 40ml-100ml (100mg pack size), and 0.4-1ml (1mg pack size). Antibody concentrations in the range 1-4mg/ml are therefore ideal. However, concentrations and volumes outside these suggested limits have also yielded excellent conjugates. For any new antibody, optimization of the ratio of antibody to PerCP-Cy5.5 is often worthwhile.
The above advice should, on average, yield an antibody:PerCP/Cy5.5 ratio of 1:1. However please bear in mind that this is an estimate of the expected molar ratio and we cannot guarantee that each antibody will be conjugated to exactly one tandem dye molecule as in reality there might be some variety.

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Answer

Thank you for contacting us.

Unfortunately, I am unable to tell you the number of Cy5.5 molecules. The amount of Cy5.5 per PerCP in the PerCP/Cy5.5 conjugation kit has been optimised to give excellent performance.


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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Answer

Thank you for contacting Abcam. Here are the details you requested. Ab102907 (PerCP ): Abs max=482nm, Emission Max= 677nm , Extinction coefficient (cm-1M-1)= 380,000 Ab102911(PerCP/Cy5.5): Abs max=482nm, Emission Max= 700nm , Extinction coefficient (cm-1M-1)= unknown I hope this information is helpful. Please let me know if you have any questions.

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Answer

Thank you for your response.
You could certainly use the credit for purchasing a different product which is in our catalogue.
Your credit note ID is 19429.
I am sorry that this antibody did not perform as stated on the datasheet. I have asked our accounting department to issue a credit note for you, which can be redeemed against the invoice of a future order by passing it on to your purchasing department. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. If you have questions on how to use the credit note, please contact our accounting department.
Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.
The credit note ID is for your reference only and does not automatically guarantee the credit.
I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

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Answer

Thank you for getting back to me and for providing some further information. Your co-operation is much appreciated. I understand that there is no additive in the storage buffer which could interact with the conjugation reaction. 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 kit as free of charge or a credit note which you could use in the future. Please do let me know how you wish to proceed. I look forward to hearing from you soon.

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Answer

Thank you for getting back to me and for confirming the correct product code number of the kit you are having difficulties. 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. 1) Could you please confirm the concentration of the primary antibody and how much protein was used for the conjugation reaction? 2) It would be also important to know the composition of the storage buffer of the antibody (buffer and pH, any BSA, cryoprotective or preservative)? I understand from your e-mail that the primary antibody has been successfully conjugated to other fluorochromes. Would you be so kind to confirm if for this purpose EasyLink conjugation kits were used as well? I look forward to hearing from you soon.

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