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Abcam's PE / R-Phycoerythrin Conjugation Kit provides a simple and quick process to conjugate your primary antibodies with R-PE. R-PE is an orange fluorescent protein which has excitation wavelengths of 496 nm, 535 nm and 566 nm and an emission wavelength of 575 nm.
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.
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.
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 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 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.
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. 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 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 (ab102778 or ab102784). (Note: Unusually, for an amine, Tris has little effect on conjugation efficiency as long as the concentration is 20mM or less).
|Components||300 µg||1 mg||30 µg||60 µg|
|Modifier reagent||1 vial||1 vial||1 vial||1 vial|
|Quencher reagent||1 vial||1 vial||1 vial||1 vial|
|R-PE mix||3 x 100µg||1 x 1mg||3 x 10µg||1 x 100µg|
Flow cytometry histogram showing integrin beta-3 positive population of platelets from wildtype and knockout mice. Integrin beta-3 antibody was conjugated using EasyLink R-Phycoerythrin conjugation kit (ab102918). Flow cytometry was performed using platelets from wild type and integrin beta-3 knockout mice. Mice that expressed beta-3 (shown in red) had a clear shift in FL-2 fluorescence over beta-3 knockout mice (shown in black).
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"