Three tips to successfully conjugate your own antibody

Wed, 02/10/2016 - 10:24

Conjugated antibodies are essential research tools for countless cell and molecular assays. While much of the time a researcher’s needs can be met with the wide variety of commercially available antibody conjugates, some applications may call for a unique reagent. For this reason some scientists opt for the flexibility of conjugating their own antibodies. While this approach may seem daunting, the availability of antibody labeling kits like the Novus Lightning-Link Kit have simplified the conjugation process and reduced the time needed to generate your own high quality antibody reagents. The Lightning-Link reagent kit allows you to efficiently label your antibody in just 30 seconds. Whether you are simply trying to save your lab a bit of money or synthesizing a unique reagent with no commercial alternatives, take into account the following advice to ensure success:

1. Starting Material
The success of labeling your antibody depends on the quality and purity of your starting material. Make sure there is sufficient starting sample and that it is stored at a high enough concentration according to kit recommendations. The storage buffer should be amine-free (e.g. PBS, HEPES, or MOPS) and free of components such as BSA, sodium azide, glycine, or tris, as they can quench the labeling reaction. AbSelect Purification kits are available to quickly and simply purify antibodies in preparation for labeling. Additionally, the AbSelect Serum Purification kit is designed to purify crude antibody formulations such as serum or ascites fluid.

2. Choose a Label
The label you choose will depend on the desired application. The properties of the various fluorophores, such as brightness or photobleaching rates, make them better suited for flow cytometry than fluorescent microscopy and vice versa. Choose dyes whose spectra won’t interfere with the others you plan to use. Additionally, be sure to choose labels that are compatible with the instruments and filter sets available to you. Lastly, detecting low level targets by fluorescent microscopy may require the use of an amplification step using biotin conjugated antibodies. Biotin can be readily visualized with streptavidin-fluorophore conjugates and can increase the sensitivity of your assay.

3. Controls
Always test and characterize the newly generated antibody. Check for proper labeling by determining the dye:protein (F/P) ratio. The labeled antibodies should be compared to unlabeled ones in applications like westerns or immunofluorescence to ensure their specificity and staining patterns have not been altered.

With some practice, conjugating your own antibodies can be quite simple. However, if you would rather, the experienced scientists at Novus can deliver consistent and reliable Custom Antibody Labeling on a small scale or in bulk. 

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