Overview

  • Product name
    Streptavidin Conjugation Kit
  • Product overview

    Streptavidin Conjugation Kit ab102921 uses a simple and quick process to conjugate an antibody to Streptavidin. It can also be used to conjugate other proteins or peptides.


    To conjugate an antibody to Streptavidin 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 Streptavidin.

  • Notes

    Amount and volume of antibody for conjugation to Streptavidin

     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 are not suitable for conjugation.

  • Tested applications
    Suitable for: Conjugationmore details

Properties

Applications

Our Abpromise guarantee covers the use of ab102921 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:
  • Lopes FB  et al. Membrane nanoclusters of Fc?RI segregate from inhibitory SIRPa upon activation of human macrophages. J Cell Biol 216:1123-1141 (2017). Read more (PubMed: 28289091) »
  • Zarnegar MA  et al. Targeted chromatin ligation, a robust epigenetic profiling technique for small cell numbers. Nucleic Acids Res 45:e153 (2017). Read more (PubMed: 28973448) »
See all 4 Publications for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Answer

Thank you for contacting us.

With regard to the number of biotins, each antibody will be conjugated with multiple biotins, getting up to 6 or 8 biotins per antibody is feasible. It is important to remember that you only need one biotin attached to enable the biotinylated antibody to bind to streptavidin. There are assays available to measure biotinylation. However, in this case they will not be suitable as there is no separation step post conjugation.

With regard to Streptavidin, you can only confirm the streptavidin is attached to the antibody by size. The easiest way is to run an ID gel and measure the molecular weight. An antibody is 160KD and streptavidin is 53KD. A conjugate will therefore by 160 + 53KD (one strep) or 160 + 106 (two streps) or 160 + 159KD (3 streps). In reality you will get a mixture of all three. Also you must run a non-reducing gel otherwise you will get a mess due to all the possible combinations as both proteins are composed of subunits.

You can control the molar ratio by adding different amounts of antibody. This will guide the actual labelling to a desired ratio. For example, adding the following amounts of antibody will give different molar ratios:

100ug of antibody to 100ug of strep = 3 streps on each antibody

150ug of antibody to 100ug of strep = 2 streps on each antibody

300ug of antibody to 100ug of strep = 1 strep on each antibody

This is only a guide and it will not be absolute, as no reaction is 100% efficient.

As for the question “Is it a robust process that would conjugate all antibodies (polyclonals and monoclonals) in a consistent way?” If the same antibody, either monoclonal or polyclonal, is used in the same conditions (buffers, incubations times, etc) the results will be very reproducible. If labeling is carried out with different antibodies then there might some variation between results.

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 your inquiry. I am sorry to hear that ab102921 did not deliver optimal results for you. I recommend to use the kit as instructed with a maximum of 100ul and 100ug of antibody. These kits are optimized for this specific protocol. There is the option of concentrating the antibody in order to be able to use 100ug for the reaction while not exceeding the maximum volume. I can recommend ab102778 for this. Click here (or use the following: https://www.abcam.com/index.html?datasheet=102778). The Easy Link Kits work by targeting 'available' amines on the antibody. They will also label proteins etc that have amines. This is the reason not to use more volume as stated and to use purified antibody. Otherwise the streptavidin will label the proteins in the buffer instead of the antibodies. Many antibodies come in buffers that contain additional protein to keep the antibody stable. I suggest to purify the antibody in this case. I can recommend ab102784 for purification. Click here (or use the following: https://www.abcam.com/index.html?datasheet=102784). I hope this information is helpful. Please do not hesitate to contact me again with any further questions.

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