NovaFluor™ Blue 725 Conjugation Kit, 100 μg - FAQs

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.

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The procedure is carried out over three days but all steps that require an overnight incubation can be allowed to rest longer before moving on. The antibody activation on day 1 is time sensitive and should not be allowed to proceed longer than 1 hr. After mixing the activated antibody with NovaFluor Linker, the antibody is incubated overnight at room temperature but following this incubation, it can be stored at 4 degrees C until ready to move on to the ammonium sulfate precipitation steps outlined on Day 2. Also, after performing the ammonium sulfate precipitation, the antibody-NovaFluor Linker conjugate can be stored in 0.09% NaN3 at 4 degrees C for up to 6 months until ready to combine with the NovaFluor label.

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Yes, NovaFluor conjugation kits will still work if you have less than 100 µg of antibody; however, we do not recommend proceeding if you have <70 µg of antibody since all included reagents are optimized for 100 µg of antibody. Adding less antibody will result in a greater degree of labelling by the NovaFluor Linker. It is best to adjust the volumes for the ammonium precipitation steps (day 2) and the final resuspension proportionally to the initial volume of antibody used.

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No. Due to the critical nature of the Zeba spin columns to this procedure, a 1x100 µg NovaFluor conjugation kit can only accommodate one antibody.

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If the antibody is only available at a lower concentration, it can be concentrated using spin columns, such as our Pierce Protein Concentrator PES, 50K MWCO, 0.5 mL (Cat. No. 88504). If your antibody is 0.5 mg/mL or greater, then the antibody/protein loss should be minimal (<5%) and can be accomplished with a short spin (2-3 mins). However, if the antibody is more dilute (<0.5 mg/mL), then you should anticipate more protein loss and you may want to purchase additional antibody to counteract any loss so that you have a total of 100 µg of antibody to label.

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We recommend ordering your antibody at 1 mg/mL or greater since the conjugation reaction is formulated for 1 mg/mL antibody concentration and will be less efficient otherwise. The antibody should be free of BSA or other stabilizing proteins since these will also be labeled and will likely increase background and/or decrease the efficiency of labeling. To ensure reproducibility, the procedure begins with a buffer exchange into our optimized Reaction Buffer. This means that your antibody can be sourced in any buffer and with any buffer additives <1 kDa, as these will be efficiently removed by the Zeba desalting resin.

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