EasyLink B-Phycoerythrin Conjugation Kit (3 x 100µg B-PE) (ab102871)
- Product nameEasyLink B-Phycoerythrin Conjugation Kit (3 x 100µg B-PE)See all B-Phycoerythrin kits ...
- Product overview
EasyLink B-Phycoerythrin Conjugation Kit provides a simple and quick process to conjugate your primary antibodies with B-PE. B-PE is an orange fluorescent protein which has an excitation wavelength of 546nm and an emission wavelength of 575nm.
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 3 labeling reactions, each up to 100µg of antibody.
Amount and volume of antibody
In view of the large size of PE (240kDa), the amount of antibody used in a labeling reaction must always be less than the amount of EL-PE, in order that the PE is in a slight molar excess. The best ratio for any new antibody reagent must be determined by experimentation but 50-60µg of IgG antibody for every 100µg of EL-PE usually gives optimal results. 60µg of antibody corresponds to an Antibody:PE molar ratio of 1:1.The volume in which the antibody is added ideally should be around 40µl. Where the concentration of antibody is relatively low, and where it is impractical to concentrate the antibody, up to twice the volume stated above may be added without any significant loss in conjugation efficiency.
- Before you add antibody to the EL-B-PE mix, add 10µl of EL-Modifier reagent for each 100µl of antibody to be labeled. Mix gently.
- Remove the screw cap from the vial of EL-B-PE 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 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.
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 (ab102778 or ab102784). (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 300 µg EL-BPE mix 3 vials EL-Modifier reagent 1 vial EL-Quencher reagent 1 vial
- Research Areas
- B PEPE
Our Abpromise guarantee covers the use of ab102871 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
EasyLink B-Phycoerythrin Conjugation Kit (3 x 100µg B-PE) images
This illustration demonstrates a general procedure and will slightly vary dependent on the conjugate used.
References for EasyLink B-Phycoerythrin Conjugation Kit (3 x 100µg B-PE) (ab102871)
ab102871 has not yet been referenced specifically in any publications.