NovaFluor™ Conjugation Kit
Reveal what you are missing with clean emitting fluorophores, conjugated to your antibodies.
The NovaFluor™ Conjugation Kit provides a simple workflow to conjugate each of our 3 NovaBlue™ and 3 NovaYellow™ labels to your antibodies with just 3 pipetting steps. With our fluorescence-by-design Phiton platform, you will be able to add more parameters to your current panels in discovery research, and resolve stained populations like never before thanks to our labels’ clean emission (near-zero cross excitation and “silent in the violet”). The consistency and stability of our labels is unmatched, and enables you to rapidly scale from discovery to screening.
Our new Wave 2 Conjugation kits adds another 13 NovaFluors™ to the range. Due for release in October 2020, we are taking pre-orders now!
Go beyond the current limitations and compromises of PE and PE-tandem dyes, resolve new cell populations, and use the full capabilities of your blue and yellow/green lasers. The advances provided by the NovaFluor™ Conjugation Kit, from only 60 seconds of hands on time for conjugation to our best-in-class block enable new colors and new biology in your laboratory right now.
Resolve what you have been missing:
- Increase the number of parameters on conventional and spectral flow cytometers with clean and bright labels
- “Stack” and utilize the capabilities of your blue and yellow/green lasers simultaneously
- Resolve populations with limited/near-zero cross excitation and the lowest spread performance of any blue and yellow-green excited fluorescent labels
- Conjugate 3 blue labels and 3 yellow-green labels to your antibodies with just 3 pipetting steps and a well characterized degree-of-labeling
Consistently amazing discovery with these Conjugation Kit features:
- Rapid, hands-off conjugation for 10μg – 100μg of antibody per label
- Simple workflow
- Optimized blocking solution for Flow Cytometry applications
- Available with Phiton technology
NovaFluorTM Conjugation Protocol
The NovaFluor Conjugation Kit provides a simple workflow to conjugate each of our NovaFluor labels to your antibodies with only 60 seconds of hands-on time.
FAQs for Phitonex NovaFluorTM Conjugation Kit
- What antibody concentrations and formats are suitable for the NovaFluor conjugation kit? Are there any restrictions?
Answer: 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 may also be
labeled and would likely increase background and/or decrease the efficiency of labeling. Compatible and incompatible buffer conditions are
listed in the table included with the conjugation kit protocol.
- What if my antibody is only available at a concentration below 1 mg/mL? Can I still use the conjugation kit?
Answer: If the antibody is only available at a lower concentration, then it can be concentrated using spin columns, such as the Pierce Protein
Concentrator PES, 50K MWCO, 0.5 mL (ThermoFisher catalog #88504) or the Amicon 50K concentrator (Sigma catalog #UFC500308). 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
minutes). However, if the antibody is more dilute (<0.5 mg/mL), then you should anticipate more protein loss and may want to purchase
additional antibody to counteract any loss so that you have a total of 100 ug of antibody to label.
- Can I label more than one antibody with a 1×100 ug conjugation kit?
Answer: This is possible in theory but has not been tested. Since the linker comes lyophilized, both antibodies would have to be available at the time of labeling (i.e. you could not split the kit and use half at one time and the other half later). One approach would be to dissolve the linker in a
minimal volume of PBS and divide the linker in half prior to Step 1 (“NovaFluor Linker Attachment to the Antibody”). In addition, you may lose
more antibody during the precipitation in Step 2 (“Removal of Excess NovaFluor Linker from DNA-Conjugated Antibody”). By using less
antibody in Step 2, the resulting pellet will be smaller, more difficult to see, and more susceptible to loss prior to labeling with the NovaFluor.
- If I want to use less antibody in Step 1, can I skip Step 2 and just anneal the NovaFluor to the Oligo-Conjugated Antibody
in Step 3?
Answer: We do not recommend this because the NovaFluor could hybridize with the free DNA linker rather than the oligo DNA linker attached to the
antibody. As a result, the efficiency of the NovaFluor labeling of your antibody will likely be decreased, and lead to a lower MFI.
- Can I complete Step 1 (or Step 2) and then stop, or do I need to complete the whole procedure at one time?
Answer: Step 1 can be completed and stored until ready for Steps 2 and 3. Step 2 is required to remove excess DNA linker prior to NovaFluor labeling in Step 3. The NovaFluor conjugation kit process can be stopped and is stable after completing Step 1 or Step 2.
If long term storage is required (prior to completion of all steps of the conjugation reaction), sodium azide can be added (no more than 0.09%
final concentration is recommended).
- In Step 2 (“Removal of Excess NovaFluor Linker from the DNA-conjugated antibody”), can you provide more information about
how I should orient my tube and describe the appearance of the pellet following precipitation?
Answer: We have included a figure (see below) to show how the pellet appears following the precipitation step. Prior to centrifugation in Step 2.3, we recommend orienting your microcentrifuge tube so that the hinge of the cap is arranged toward the outside of the centrifuge to ensure that the location of the pellet will be clear (or mark with a sharpie the outside orientation of the tube). Make sure to keep the supernatant from the precipitation at 4oC until the conjugation is complete in case the antibody does not precipitate.
- In Step 2, I see there is just 1 round of precipitation recommended. Weren’t there 2 rounds of precipitation in the original NovaFluor conjugation protocol? Answer: Yes, in the original version of the NovaFluor conjugation protocol in Step 2, there were 2 rounds of precipitation. However, we found in practice that 1 round of precipitation was sufficient to remove most of the excess NovaFluor Linker (~85%). Notably, the second round of precipitation does not substantially enhance the resulting median fluorescence intensity (MFI) of the NovaFluor conjugated antibody after Step 3 (see Figure 2 below). In addition, following one round of precipitation at the 100 μg scale, the pellet appears compact and white. However, following the second precipitation, the pellet is oblong and translucent and thus harder to see and in some cases recover. Since this second precipitation step may lead to a greater loss of antibody, we no longer recommend two rounds of precipitation in the NovaFluor Conjugation protocol.
- In Step 3, (“Annealing of NovaFluor to Oligo-Conjugated Antibody”), it looks like the annealing step has been simplified. What is the rationale for this change? Answer: We have found that there is no substantial difference in the resulting MFI of the NovaFluor antibody conjugate when assembled from room temperature to 4ºC for four hours versus the previous process of annealing from 50ºC to 4ºC over four hours.
- How should NovaFluor-antibody conjugates be stored? Answer: New conjugates should be stored at 4ºC and protected from light. If desired, NovaFluor-antibody conjugates can be stored with 0.09% sodium azide. DO NOT store NovaFluor-antibody conjugates at -80ºC or -20ºC.