Overview

  • Product name
    Cy5® Conjugation Kit (Fast)
  • Product overview

    Cy5® Fast Conjugation Kit (ab188288) uses a simple and quick process to conjugate an antibody to Cy5. It can also be used to conjugate other proteins or peptides.


    To conjugate an antibody to Cy5 using this kit:
    - add modifier to antibody and incubate for 15 min
    - add quencher and incubate for 5 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 Cy5.

  • Notes

    Amount and volume of antibody for conjugation to Cy5

     Kit size   Recommended 
    amount of antibody
    1 
    Maximum 
    amount of antibody
    Maximum antibody 
    volume2
    30 µg   3 x 10 µg  3 x 20 µg 3 x 10 µL
    300 µg   3 x 100 µg  3 x 200 µg 3 x 100 µL
    1 mg   1 mg 2 mg 1 mL

    Using the maximum amount of antibody may result in less labelling per antibody. 

    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 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 ab188288 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 concentration.

Images

Protocols

References

This product has been referenced in:
  • Rizvi AH  et al. Single-cell topological RNA-seq analysis reveals insights into cellular differentiation and development. Nat Biotechnol 35:551-560 (2017). Read more (PubMed: 28459448) »
See 1 Publication for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Answer

Only the BSA seems to be a problem, its concentration should be 0.1%-0.5%. The ideal way is to remove BSA, we have kit for this https://www.abcam.com/BSA-Removal-Kit-ab173231.html. Alternatively you can give it a go however it might affect the efficiency of the kit. Please check the information on booklet;

10.2 Common non-buffering salts (e.g. sodium chloride), chelating agents (e.g.EDTA), and sugars have no effect on conjugation efficiency. Azide (0.02 to 0.1%) and BSA (0.1 to 0.5%) have little or no effect. Glycerol up to 50% has no effect.

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Answer

M13 bacteriophages have not been tested with our labeling kits. However, as long as it contains some primary amines which are available for labeling, then there should be no reason why this would not work. As for antibodies, it is important that the buffer does not contain any free amines or other unsuitable substances. Please see below for details of (un)compatible formulations:


Additives such as salts (e.g NaCl), sugars (e.g. sucrose) and chelators (e.g. EDTA) have no effect on the labeling reaction. We recommend avoiding nucleophiles such as amino acids (e.g. glycine), blockers (e.g. ethanolamine) and thiols (DTT, mercaptoethanol) that could interfere with the conjugation reaction.

Compatible additives:
up to 20mM Tris
up to 0.1% BSA (BSA up-to 1% will work but yield lower quality conjugates)
up to 0.1% gelatin
up to 0.1% sodium azide
PBS pH6.5-8.5
up to 50% glycerol
0.15M sodium chloride
50mM HEPES
0.02M potassium phosphate
0.001% Tween
Proclin 300
5% Trehalose


Incompatibile additives:
Tris above 20mM
Urea
50mM Imidazole
Glycine
Ethanolamine
DTT
Mercaptoethanol


Finally, the M13 bacteriophage will be a different molecular weight to that of an antibody, and the protocol will therefore have to be adjusted to allow for the size difference. It is difficult for us to provide any advice, as I do not know the MW of the bacteriophage, but I would generally recommend that you carry out a titration of different concentrations, in order to determine the best set-up for your assay.

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