Overview
- Product nameEasyLink Alkaline phosphatase Conjugation Kit (1 x 1mg AP)See all Alkaline phosphatase kits ...
- Product overview
EasyLink 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.
This kit is sufficient for 1 labeling reaction of 1mg of antibody.
- Notes
Amount and volume of antibody
The recommended amount of antibody to be used for labeling is 1mg. The volume in which the antibody is added ideally should be in the range 0.4-1ml. 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
- Before you add antibody to the EL-AP mix, add 100µl of EL-Modifier reagent for 1ml of antibody to be labeled. Mix gently.
- Remove the screw cap from the vial of EL-AP mix and pipette the antibody sample (with added EL-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 EL-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 EL-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 EL-modifier for each 10µl of antibody. Excess EL-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 EasyLink 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). (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.
- Tested applicationsConjugation more details
Properties
- Storage instructionsStore at -20°C. Please refer to protocols.
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Components Identifier 1 mg EL-AP-Mix — EL-Modifier reagent — EL-Quencher reagent — - Research Areas
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Alternative names
- AP
Applications
Our Abpromise guarantee covers the use of ab102848 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Notes |
|---|---|
| Conjugation | Conjugation |
EasyLink Alkaline phosphatase Conjugation Kit (1 x 1mg AP) images
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Conjugation - EasyLink Alkaline phosphatase Conjugation Kit (1 x 1mg AP) (ab102848)This illustration demonstrates a general procedure and will slightly vary dependent on the conjugate used.
References for EasyLink Alkaline phosphatase Conjugation Kit (1 x 1mg AP) (ab102848)
ab102848 has not yet been referenced specifically in any publications.
