Jun 09, 2026
Site-Specific Antibody Labeling with FlexAble Kits
- Marina Able1
- 1Proteintech Germany GmbH
- Proteintech
Protocol Citation: Marina Able 2026. Site-Specific Antibody Labeling with FlexAble Kits. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp48w5gzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working.
Created: June 05, 2026
Last Modified: June 09, 2026
Protocol Integer ID: 318613
Keywords: Antibody labeling, Antibody conjugation, Antibody Dye Conjugation, Multiplex immunofluorescence, Antibody Enzyme Conjugation, Site-specific Conjugation, Site-specific Labeling, Antibody Labeling Kit, specific antibody labeling with flexable kit, specific antibody labeling, direct labeling of primary antibody, fc part of primary antibody, labeled primary antibody, several antibody candidate, compatible with primary antibody, labeled antibody, rapid antibody, specific antibody clone, primary antibody, antibody buffer, use of flexable, flexable labeling, flexable oligo, immunofluorescence imaging, specific attachment of fluorophore, oligonucleotide, flexable, flexable kit, labeling technology, immunostaining, immunohistochemistry, fluorophore, factor for immunostaining, flow cytometry
Abstract
The availability of directly labeled primary antibodies can ben a limiting factor for immunostaining, particularly when working with less common host species, special targets, or specific antibody clones. Direct labeling of primary antibodies may also be desirable to enable same-species multiplexing or to simplify and accelerate workflows.
This protocol describes the use of FlexAble, a simple and rapid antibody-labeling technology that enables site-specific attachment of fluorophores, enzymes, biotin, or oligonucleotides (FlexAble Oligo-Ready) to the Fc part of primary antibodies at micro- to milligram scale. FlexAble labeling can be completed in just 15 minutes, without the need for specialized equipment or conjugation expertise. The technology is compatible with primary antibodies from any supplier and works in virtually any antibody buffer, as common additives such as glycerol do not interfere with the reaction.
FlexAble-labeled antibodies have been validated in a wide range of downstream applications, including (cyclic) immunofluorescence imaging, immunohistochemistry, flow cytometry, and Western blotting. In addition, FlexAble provides a convenient way to screen several antibody candidates due to its low input material requirement and fast workflow.
Materials
- FlexAble Antibody Labeling Kit (Proteintech)
| A | B | C | D | E | F | G | H | I | J | |
| CoraLite Plus 405 | CoraLite Plus 488 | FITC Plus | CoraLite Plus 555 | CoraLite Plus 594 | CoraLite Plus 647 | CoraLite Plus 750 | HRP | Biotin | ||
| Rabbit IgG | KFA506 | KFA501 | KFA508 | KFA502 | KFA509 | KFA503 | KFA504 | KFA005 | KFA007 | |
| Mouse IgG1 | KFA526 | KFA521 | KFA528 | KFA522 | KFA529 | KFA523 | KFA524 | KFA025 | KFA027 | |
| Mouse IgG2a | KFA546 | KFA541 | KFA548 | KFA542 | KFA549 | KFA543 | KFA544 | KFA045 | KFA047 | |
| Mouse IgG2b | KFA566 | KFA561 | KFA568 | KFA562 | KFA569 | KFA563 | KFA564 | KFA065 | KFA067 | |
| Human IgG | KFA608 | KFA604 | KFA609 | KFA605 | KFA612 | KFA606 | KFA607 | KFA110 | KFA111 | |
| Rat Kappa light chain | KFA621 | KFA626 | KFA622 | KFA623 | KFA128 |
Overview of Species and Conjugate Options
- Unconjugated antibody you'd like to label
- Eppendorf tubes
- Optional (for FlexAble Biotin Kits only): Streptavidin dyes
Before start
Before you begin, equilibrate all reagents to room temperature.
General Notes
Workflow overview
This protocol is for labeling of 0.5 µg of primary antibody. Scale up proportionately to label larger quantities of primary antibody.
Protocol
Before you begin, equilibrate all reagents to room temperature.
Combine 0.5 µg of primary antibody with 1 µL of FlexLinker. Add FlexBuffer to bring total volume to 8 µL.
Mix gently and incubate for 5 minutes at room temperature in the dark.
Add 2 µL of FlexQuencher.
Mix gently and incubate for 5 minutes at room temperature in the dark.
The antibody is now ready to be used.
Notes on Multiplexing
Primary antibodies of different species can be combined in the same tube and labeled simultaneously using corresponding species-specific FlexLinkers. Scale up the reagent volumes appropriately based on the amount of primary antibody to be labeled.
For primary antibodies of the same species, label each antibody individually. Once the primary antibodies are labeled, combine and use in your desired application.
Antibodies labeled with the FlexAble kit can be used alongside pre-conjugated primary antibodies.
Special notes for FlexAble Biotin Kits
FlexAble Antibody Labeling (for FlexAble Biotin Kits)
Use the standard workflow described above to label your antibodies with Biotin. Add 1 µg of Streptavidin dye together with the FlexQuencher in Step 5. All other steps are identical.
Immunostaining Workflow (for FlexAble Biotin Kits)
Fix, permeabilize and block cells according to your standard application protocol.
Block cells with Streptavidin Blocking blocking solution (not included with FlexAble or FlexAble 2.0 kits). Incubate for 15 min at room temperature and wash 3x with PBS.
Incubate samples with prepared FlexAble labeled antibodies in your assay buffer for 1 h at room temperature. Then, wash 3x with PBS.
If desired, add DAPI.
For immunofluorescence of tissues or cells, add mouting media and a coverslip before imaging. For flow cytometry, your samples can be resuspended in assay buffer after step 13 and are ready to be used.