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Abcan's APC/Cy5.5 Conjugation Kit provides a simple and quick process to conjugate your primary antibodies with APC/Cy5.5. APC/Cy5.5 is a tandem conjugate: APC (energy donor) has an excitation wavelength of 650nm and Cy5.5 (energy acceptor) has an emission wavelength of 694nm.
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 best ratio for any new antibody reagent must be determined by experimentation but 100-150µg of IgG antibody for every 100µg of APC/Cy5.5 usually gives optimal results. The 150µg quantity corresponds to an Antibody:APC/Cy5.5 molar ratio of about 1:1.
The volume in which the antibody is added ideally should be between 40 and 100µl.
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 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.
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||30 µg||1 mg||100 µg||300 µg|
|APC-CY5.5 Mix||3 vials||1 vial||1 vial||3 vials|
|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 ab102855 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.|
This illustration demonstrates a general procedure and will slightly vary dependent on the conjugate used.
ab102855 has not yet been referenced specifically in any publications.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"