Alkaline phosphatase Conjugation Kit (ab102850)

Overview

  • Product nameAlkaline phosphatase Conjugation Kit
  • Product overview

    Abcam's Alkaline Phosphatase Conjugation Kit provides a simple and quick process to conjugate your primary antibodies with Alkaline phosphatase.

    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

    The recommended amount of antibody to be used for labeling is 100µg (on each reaction). The volume in which the antibody is added ideally should be in the range 40-100µl. 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 AP is often worthwhile.

    Protocol

    1. Before you add antibody to the AP mix, add 10µl of Modifier reagent for each 100µl of antibody to be labeled. Mix gently.
    • Remove the screw cap from the vial of AP 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 at room temperature (20-25°C). Alternatively, and sometimes 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 (ab102778 or ab102784). (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.

     

    The antibody to be labelled should be purified, in an appropriate buffer for conjugation and at a suitable concentration, as described in the protocol booklet. If not, consider using our antibody purification and concentration kits.

     

  • Tested applicationsSuitable for: Conjugationmore details

Properties

  • Alternative names
    • AP

Applications

Our Abpromise guarantee covers the use of ab102850 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.

Alkaline phosphatase Conjugation Kit images

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

Protocols

References for Alkaline phosphatase Conjugation Kit (ab102850)

ab102850 has not yet been referenced specifically in any publications.

Product Wall

EasyLink AP Kit (ab102848)

Good Excellent 5/5 (Ease of Use)
Abreviews
The kit is easy to use and achieve its purpose.
I have one suggestion: to add some kind of detection, after conjugation is finished.
This will able the customer to detect the success of AP conjugation, before using the AP conjugated protein in the intended protocol.

Tested by ELISA
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Abcam user community

Verified customer

Submitted May 02 2014

The conjugation protocol requires the initial antibody volume be diluted 20% during the conjugation reaction. 1/10 antibody volume of EL-Modifier is added at the beginning of the reaction and antibody 1/10 volume of EL-Quencher is added at the conclusi...

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Thank you for your reply and for providing that information.

I believe the cause of the problem is the purity of the rabbit antibody. As the antibody is purified using ammonium sulphate and not Protein A/G, then the sample of antibody is not ...

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Thank you for contacting Abcam yesterday.

I have talked to the lab about the issue you have been having and our kits are designed primarily for labeling IgGs; the failed conjugation would not have been because rabbit IgG was used. They have ...

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Thank you for contacting Abcam. The 4mM phosphate buffer concentration will work for ab102850. Also the antibody must be purified and at a concentration of 1mg/ml and additives such as BSA and azide can reduce conjugation efficiency.   If there is anyt...

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