Invitrogen™ NovaFluor™ Antibody Conjugation Kits

Catalog No.	Label or Dye	Product Type	Emission	Quantity
K06T04L026	NovaFluor™ Blue 760	Conjugation Kit	760 nm	100 μg
K06T04L008	NovaFluor™ Blue 510	Conjugation Kit	510 nm	100 μg
K06T04L002	NovaFluor™ Blue 530	Conjugation Kit	530 nm	100 μg
K06T04L010	NovaFluor™ Blue 585	Conjugation Kit	585 nm	100 μg
K06T04L003	NovaFluor™ Blue 610/30S	Conjugation Kit	610 nm	100 μg
K06T04L004	NovaFluor™ Blue 660/40S	Conjugation Kit	660 nm	100 μg
K06T04L021	NovaFluor™ Blue 690	Conjugation Kit	690 nm	100 μg
K06T04L012	NovaFluor™ Blue 660/120S	Conjugation Kit	660 nm	100 μg
K06T04L017	NovaFluor™ Red 660	Conjugation Kit	660 nm	100 μg
K06T04L018	NovaFluor™ Red 685	Conjugation Kit	685 nm	100 μg
K06T04L019	NovaFluor™ Red 700	Conjugation Kit	700 nm	100 μg
K06T04L020	NovaFluor™ Red 710	Conjugation Kit	710 nm	100 μg
K06T04L023	NovaFluor™ Red 725	Conjugation Kit	725 nm	100 μg
K06T04L024	NovaFluor™ Red 755	Conjugation Kit	755 nm	100 μg
K06T04L005	NovaFluor™ Yellow 570	Conjugation Kit	570 nm	100 μg
K06T04L013	NovaFluor™ Yellow 590	Conjugation Kit	590 nm	100 μg
K06T04L006	NovaFluor™ Yellow 610	Conjugation Kit	610 nm	100 μg
K06T04L007	NovaFluor™ Yellow 660	Conjugation Kit	660 nm	100 μg
K06T04L014	NovaFluor™ Yellow 690	Conjugation Kit	690 nm	100 μg
K06T04L015	NovaFluor™ Yellow 700	Conjugation Kit	700 nm	100 μg
K06T04L016	NovaFluor™ Yellow 730	Conjugation Kit	730 nm	100 μg
K06T04L022	NovaFluor™ Yellow 755	Conjugation Kit	755 nm	100 μg

Catalog No. K06T04L026 Supplier Invitrogen™ Supplier No. K06T04L026

Description

Get the most out of your panel design by conjugating antibodies to NovaFluor dyes specifically designed for spectral hygiene to have narrow emission spectra and minimal cross-laser excitation.

NovaFluor Conjugation Kits provide a simple workflow to conjugate a blue, yellow-green or red excitable fluorophore to your antibody of choice in just 3 easy steps. The fluorescence-by-design Phiton platform enables the addition of parameters to your current panels in discovery research and resolves stained populations with near-zero cross excitation, with a lack of emission in the violet channels that plague other fluorophores.

NovaFluor Conjugation Kit provides features including:

• A clean and bright antibody label to increase the number of parameters on conventional and spectral flow cytometers
• Resolve populations with limited/near-zero cross excitation and the lowest spread performance of any blue, yellow-green or red excited fluorescent labels
• Simple 3-step protocol, with 60 sec. of hands-on time for conjugating 10–100 μg of antibody per label
• Up to 70% less excitation when compared to PE tandems, resulting in decreased spillover and higher resolution

3-step conjugation method

• Attach NovaFluor Linker to antibody of choice
• Remove excess NovaFluor Linker from DNA-conjugate antibody
• Anneal NovaFluor to oligo-conjugated antibody

Specifications

• For Use With (Application): Flow Cytometry
• Quantity: 100 μg
• Product Type: Conjugation Kit
• Label or Dye: NovaFluor™ Blue 760
• Product Line: NovaFluor
• Emission: 760 nm

Frequently Asked Questions (FAQs)

What is the NovaFluor dye structure and how does this impact the NovaFluor antibody conjugation workflow?

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.

Can I use NovaFluor antibody conjugation kits for conjugation of antibodies targeting intracellular epitopes?

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.

Can I label my antibody with more than one NovaFluor label if I purchased more than one NovaFluor conjugation kit?

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.

How should I store NovaFluor-antibody conjugates?

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.

For the NovaFluor conjugation kits, in Step 16 of the procedure, can you provide more information about how I should orient my tube and describe the appearance of the pellet following precipitation?

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.

For the NovaFluor conjugation kits, do I need to complete the procedure in the exact time course as described or are there convenient breaking points?

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.

Would NovaFluor conjugation kits work if I have less than 100 µg of starting antibody?

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.

Can I label more than one antibody with a 1x100 µg NovaFluor conjugation kit?

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.

What if my antibody is only available at a concentration below 1 mg/mL? Can I still use a NovaFluor conjugation kit?

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.

What antibody concentrations and formats should I use with NovaFluor conjugation kits? Are there any restrictions?

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.

For Research Use Only. Not for use in diagnostic procedures.