EasyLink HRP Conjugation Kit (1 x 1mg HRP) (ab102889)
- Product nameEasyLink HRP Conjugation Kit (1 x 1mg HRP)See all HRP kits ...
- Product overview
EasyLink 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.
This kit is sufficient for 1 labeling reaction of up to 4mg of antibody.
Amount and volume of antibody
The amount of antibody used for labeling ideally should 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 EL-HRP you need to add between 1-4mg of antibody. The volume of the antibody sample ideally should be up to up to 1ml. 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.
- Before you add antibody to the EL-HRP 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-HRP 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 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 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.
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. 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).
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).
Labeling of the antibody will not work if the conjugation blocks the active paratope. This situation is rare.
- Tested applicationsConjugation more details
- Storage instructionsStore at -20°C. Please refer to protocols.
Components 1 mg EL-HRP mix 1 vial EL-Modifier reagent 1 vial EL-Quencher reagent 1 vial
- Research Areas
- Horseradish peroxidase
Our Abpromise guarantee covers the use of ab102889 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
EasyLink HRP Conjugation Kit (1 x 1mg HRP) images
This illustration demonstrates a general procedure and will slightly vary dependent on the conjugate used.
References for EasyLink HRP Conjugation Kit (1 x 1mg HRP) (ab102889)
ab102889 has not yet been referenced specifically in any publications.