概述

  • 产品名称HRP偶联试剂盒
  • 产品概述

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

    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.

     

  • 说明

    Amount and volume of antibody

    The amount of antibody used for labeling should ideally correspond to molar ratios between 1:4 and 1:1 Antibody to HRP. Taking account of the molecular weights (160,000 versus 40,000), this means for that for 1mg HRP you need to add between 1-4mg of antibody. The volume of the antibody sample should ideally be up to  100µl. Antibody concentrations in the range 0.5-5mg/ml generally give optimal results, but concentrations and volumes outside these suggested ranges have also yielded active conjugates.

    Protocol

    • Before you add antibody to the HRP mix, add 1µl of Modifier reagent for each 10µl of antibody to be labeled. Mix gently.
    • Remove the screw cap from the vial of HRP 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. 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).
    Sodium azide can interfere during the conjugation reaction and is known to irreversibly inhibit HRP. We do not recommend performing a HRP conjugation in presence of sodium azide or adding sodium azide with your HRP conjugated antibody.
    (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.

  • 经测试应用适用于: Conjugationmore details

性能

  • 别名
    • Horseradish peroxidase

应用

Our Abpromise guarantee covers the use of ab102890 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用 Ab评论 说明
Conjugation Use at an assay dependent dilution.

HRP Conjugation Kit 图像

实验方案

HRP Conjugation Kit (ab102890)参考文献

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Product Wall

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