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Abcam's PE/Cy5 Conjugation kit provides a simple and quick process to conjugate your primary antibodies with PE/Cy5. PE/Cy5 is a tandem conjugate: PE (energy donor) has an excitation wavelength of 565nm and Cy5 (energy acceptor) has an emission wavelength of 666nm.
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
In view of the large size of the PE/Cy5 label, the amount of antibody used in a labeling reaction with the tandem must always be less than the amount of PE/Cy5, in order that the PE/Cy5 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/Cy5 usually gives optimal results. 50µg of antibody corresponds to an Antibody:PE/Cy5 molar ratio of 1:1.
The volume in which the antibody is added ideally should be around 400µl. 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 (ab102776 or ab102783). (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||1 mg||100 µg||300 µg||30 µg|
|Modifier reagent||1 vial||1 vial||1 vial||1 vial|
|PE/Cy5 mix||1 vial||1 vial||3 vials||3 vials|
|Quencher reagent||1 vial||1 vial||1 vial||1 vial|
Our Abpromise guarantee covers the use of ab102893 in the following tested applications.
|Conjugation||Use at an assay dependent dilution.|
ab102893 has not yet been referenced specifically in any publications.