Table of contents

1. Choose the right conjugation kit for your application
2. What is antibody conjugation and how does it work?
3. Fluorophore selection for antibody conjugation
4. Quick and easy-to-use conjugation kits for over 45 labels
5. Degree of labeling (DOL) and F:P ratio
6. What are the benefits of Lightning-Link® antibody labeling technology?
7. Supporting downstream applications
8. FAQ

Choose the right conjugation kit for your application

Selecting the right conjugation kit depends on your assay type, detection method, and label choice. The guide below helps match your application to the most suitable labeling strategy.

This selection guide helps streamline decision-making by aligning your experimental needs with the appropriate conjugation approach.

What is antibody conjugation and how does it work?

Antibody conjugation, also known as antibody labeling, chemically links an antibody to a specific tag. When conjugated, a primary antibody directly detects the target antigen of interest, without the need for a secondary antibody.

Labels can include fluorescent dyes, enzymes or proteins and your choice or combination of labels will depend on the experimental application (multiplex IHC,  flow cytometry, LFA, others), as well as your multiplex panel.

Using directly labeled primary antibodies can save you a lot of time and simplify your experimental protocols.

Fluorophore selection for antibody conjugation

Fluorophores are one of the most widely used labels in antibody conjugation, particularly for fluorescence-based applications such as flow cytometry, immunofluorescence, and multiplex imaging.

When selecting a fluorophore, key factors to consider include:

• Excitation and emission spectra (instrument compatibility)
• Brightness and photostability
• Spectral overlap in multiplex panels
• Compatibility with your assay

Common fluorophore options include:

• Alexa Fluor® dyes (eg, AF488, AF647)
• DyLight Fluor® dyes
• FITC, Cy dyes, APC, and R-PE
• Tandem dyes for multicolor applications

Choosing the right fluorophore is critical for achieving strong signal intensity and accurate data in complex experiments. Ensure the fluorophore selection aligns with your instrument’s laser configuration and detection channels.

Quick and easy-to-use conjugation kits for over 45 labels

Conjugating primary antibodies can lead to antibody loss and take up valuable lab time. Our innovative Lightning-Link® labeling technology enables the direct labeling of antibodies or proteins, providing a straightforward solution to traditional conjugation challenges.

Our Lightning-Link® conjugation kits are easy to use and allow you to conjugate antibodies in three simple steps to more than 45 labels including fluorescent dyes (Alexa Fluor^® and DyLight Fluor^® dyes), tandem dyes, enzymes, oligonucleotides, and gold nanoparticles.

We recommend using Lightning-Link® conjugation kits with our carrier-free antibodies to maximize efficiency and minimize antibody loss.

Download the application note to see the data on the consistent performance of the Lightning-Link® kits.

Degree of labeling (DOL) and F:P ratio

The degree of labeling (DOL), or fluorophore-to-protein (F:P) ratio, describes how many label molecules are attached to each antibody. It is typically calculated by measuring absorbance of the conjugated antibody and applying standard ratio formulas.

Optimizing the DOL is important to balance the signal strength with antibody performance.

Key considerations:

• Low DOL – weaker signal
• High DOL – potential interference with antigen binding
• Optimal DOL – strong signal with preserved antibody function

DOL depends on label type, antibody concentration, and conjugation chemistry. Maintaining an appropriate ratio ensures reproducible and reliable assay results.

How does it work?

Simply pipette your antibody or biomolecule of choice into the vial of a lyophilized mixture containing the label of interest and incubate for just 15 minutes (Lightning-Link^® Fast range) or around 3 hours (Lightning-Link^® range).

​​​​The process generates reproducible conjugates with no antibody loss and no purification steps required.

What are the benefits of Lightning-Link^® antibody labeling technology?

• Simple and consistent: Conjugate your antibody in three simple steps without the risk of antibody loss from a traditional, post-conjugation purification step.

• Quick and easy: Our conjugation kits only require 30 seconds of hands-on time so you can focus on your research. Watch our short protocol video to find out more.

• Choice and flexibility: Access a broad range of labels, from fluorescent dyes and enzymes to metal isotopes and gold nanoparticles. Ready to be used with our 13,000 conjugation-ready carrier-free antibodies, giving you the ultimate flexibility to design and complete your experiment.
• Scalable and efficient: With kits that work with 10 µg-100 mg of antibody, achieve consistent conjugation whatever the scale of your experiment

Browse our most popular conjugation kits:

Enzymes: HRP | Alkaline Phosphatase
Proteins: Biotin | Streptavidin
Fluorophores: AF®488 | AF®647 | R-PE | APC | FITC | Cy3® | Cy5®
Tandem dyes: PE/Cy7® | APC/Cy7® | PE/Cy5® | PerCP/Cy5.5® | PE/Cy5.5®
Oligonucleotide: Universal
Metal Isotopes: 169Tm | 141Pr | 160Gd | 175Lu | 165Ho
Gold nanoparticles: 40 nm | 20 nm | 10 nm

Alexa Fluor^® conjugation kits

These kits facilitate easy and reliable conjugation of antibodies with Alexa Fluor® dyes, known for their bright and stable fluorescence. We offer AF488, AF555, AF647 and more.

 
Fluorescent conjugation kits

Enable conjugation of antibodies with a variety of fluorescent dyes (eg Cy dyes, ATTO, FITC, APC, and more), enhancing visualization in fluorescence-based applications. Conjugation kits also come in various tandem dyes for flow cytometry applications.

HRP and alkaline phosphatase conjugation kits

Ideal for enzyme-linked immunodetection, these kits link antibodies with horseradish peroxidase (HRP) or Alkaline Phosphatase (AP) for various assays.

Biotinylation and streptavidin conjugation kits

Kits designed for the biotinylation of antibodies that can be detected or captured using streptavidin, offering high affinity and specificity.

Other conjugation tools

Explore a variety of additional conjugation tools, including kits for labeling with gold nanoparticles, metal isotopes, latex and more.

Explore our range of fluorescent conjugated antibodies or learn how to generate reproducible conjugates with our antibody conjugation guide.

If you're interested in outsourcing antibody labeling to our team of experts, please get in touch and ask about our custom conjugation services. Contact us today to discuss your needs.

Supporting downstream applications

Antibody conjugation plays a critical role in many experimental workflows.

Conjugated antibodies are widely used in:

• Immunohistochemistry (IHC) – for tissue-based protein detection
• Flow cytometry – for multicolor cell analysis
• Lateral flow assays (LFA) – for rapid and point-of-care testing
• ELISA and immunoassays – for quantitative protein detection

Selecting the appropriate label ensures compatibility with your downstream application and improves reproducibility.

FAQ

How does Lightning-Link® conjugation work?

The labeling chemistry targets primary amines present in lysines and at the N-terminus of a protein. All antibodies have multiple free amine groups and most proteins have lysine and/or alpha-amino groups. The antibody or biomolecule simply needs to be pipetted into a vial of lyophilized mixture containing the label of interest, and incubated for around three hours (Lightning-Link® range) or 15 minutes (Lightning-Link® Fast range).

Despite the apparent simplicity of its protocol, the Lightning-Link® Conjugation process is sophisticated and quickly generates reproducible conjugates with no loss of antibody, saving you valuable time and resources.

What recovery can be expected?

With Lightning-Link® conjugation kits, the entire antibody labeling reaction is contained within one tube and there are no separation steps involved. This means that 100% of antibody is retained at the end of the conjugation process. The conjugation process does not trigger antibody aggregation, and it is carried out at a physiological pH. Once the reaction is complete, you can usually use the conjugated antibody straight away without further purification steps.

Can I conjugate the same antibody with two different Lightning-Link® labels?

No. Lightning-Link® Conjugation Kits are designed for labeling antibodies with one type of label (single labeling). – kits are only recommended for single labelling.

What is the best way to check conjugation success?

Our range of conjugation check kits allows you to confirm the conjugation of an antibody in one easy step, without the need for any specialized or costly equipment. Please note that these kits are only suitable for the qualitative verification of IgG antibodies.

Alternatively, you can test conjugation success using a preliminary experiment in the application of interest.

How do I filter out the free label from the conjugated antibody?

Lightning-Link® conjugation kits are designed to give a low level of free label at the end of the reaction. Thus, no filtration steps are required. Any remaining free label would have its reactive groups blocked by the Quencher provided in the kit, and would then be washed away during the relevant wash step of your application.

Can I use the Lightning-Link® conjugation kits with primary antibodies stored in BSA, glycerol, Tris buffer and/or preservatives?

We recommend using our carrier–free primary antibodies, as certain buffer constituents are incompatible with the labeling reaction. If your antibody does not meet these requirements, you can easily purify or concentrate your antibody using our purification and concentration kits.

Can I use the Lightning-Link® conjugation kits crude serum or tissue culture supernatant?

These kits are recommended for use with only purified antibodies or proteins.

Our guide provides more details on Labeling non-antibody proteins, peptides, and small molecules with Lightning-Link® kits

References

1. Lantz, L., Holmes, K. & Douagi, I. Conjugation of fluorochromes to monoclonal antibodies. Curr. Protoc. 3, e795 (2023).
2. Martin, C. et al. In vitro characterization and stability profiles of antibody–fluorophore conjugates derived from interchain cysteine cross-linking or lysine bioconjugation. Pharmaceuticals 12, 176 (2019).
3. McCombs, J. R. & Owen, S. C. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 17, 339–351 (2015).