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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.
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.
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 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).
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.
|Components||100 µg||1 mg||30 µg||300 µg|
|HRP mix||1 x 100µg||1 x 1mg||3 x 10µg||3 x 100µg|
|Modifier reagent||1 vial||1 vial||1 vial||1 vial|
|Quencher reagent||1 vial||1 vial||1 vial||1 vial|
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.
|Conjugation||Use at an assay dependent dilution.|
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"