NovaFluor™ Antibody Conjugation Kits

NovaFluor dyes are based on Phiton technology utilizing novel nucleic acid dye structures. The dyes work with FRET technology and have a clear benefit versus tandem dyes, as the structures are extremely stable. More information can be found on our NovaFluor website.

Due to the nucleotide structure, some adherence of the NovaFluor dyes to dead cells is expected. Good viability of the sample and inclusion of a fixable viability stain is recommended. Using nucleotide-staining viability dyes such as DAPI, PI, and 7-AAD dyes is not recommended.

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NovaFluor antibody conjugation kits can be used for conjugation of antibodies targeting intracellular epitopes. The labeling needs to be performed with 10 µL CellBlox Plus Blocking Buffer (Cat. No. C001T02F01, C001T03F01, C001T06F01) to block non-specific binding of NovaFluor labels with cells, after fixation and permeabilization.

If NovaFluor dyes are used for surface targets as well as intracellular targets, first add 5 µL CellBlox Plus Blocking Buffer during staining with the surface markers. Then, after completing the staining, washing, fixation, and permeabilization, add 10 µL CellBlox Plus Blocking Buffer again to block non-specific intracellular binding while labeling with the NovaFluor conjugates for intracellular targets.

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Yes, you can. After performing the steps on Days 1 and 2, your Antibody-NovaFluor Linker conjugate can be attached to the NovaFluor label at whatever volume you like, as long as the ratio of the Antibody-NovaFluor Linker to the NovaFluor label is kept constant.

Example: If you purchased two NovaFluor conjugation kits, one with NovaFluor Blue 610-30S and one with NovaFluor Yellow 610, use the contents of both kits to modify your two antibodies of interest (both 100 µg), completing all steps outlined for Days 1 and 2 with no modification. After resuspending your antibodies into PBS and after the second round of ammonium sulfate precipitation, you should have 100 µL of each Antibody-NovaFluor Linker conjugate. You can now divide the antibody solutions in half (50 µL each) and add half of each NovaFluor label to the divided antibody solutions (150 µL of NovaFluor label to each 50 µL of Antibody-NovaFluor Linker), giving you 200 µL total of each antibody conjugated to both NovaFluor Blue 610-30S and NovaFluor Yellow 610. After mixing, continue with the protocol and incubate overnight at 4 degrees C.

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We recommend storing new NovaFluor-antibody conjugates at 2-8 degrees C, protected from light. If desired, NovaFluor-antibody conjugates can be stored with 0.09% sodium azide. DO NOT store NovaFluor-antibody conjugates at -80 degrees C or -20 degrees C.

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We have included Figure 4 in the manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0025061_NovaFluorConjugationKit_UG.pdf) to show how the pellet appears following the precipitation step. Although the microcentrifuge tube can be centrifuged in any orientation, we recommend orienting it in the same direction for both centrifugation steps so that your pellet is in the same place (Figure 3). The pellet is often smaller after the second precipitation, so it is helpful to know where to expect it.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.