Learn the essentials of antibody conjugation with our user-friendly guide.
Updated August 2,2023.
Download our guide to antibody conjugation
Overview
- What is antibody conjugation?
- Commonly used labels
- How to conjugate antibodies?
What is antibody conjugation?
Antibody conjugation, also known as antibody labeling, is the process of linking an antibody to a specific tag or label. Most immunoassays require antibodies either directly conjugated to a specific label or detected by a conjugated secondary antibody to provide a measurable signal.
When conjugated, a primary antibody allows for the direct detection of the target antigen of interest without needing a secondary antibody. Using direct detection has pros and cons, which are explored inour antibody conjugation guide.
Antibody conjugation, also known as antibody labeling, is the process of linking an antibody to a specific tag or label. Most immunoassays require antibodies either directly conjugated to a specific label or detected by a conjugated secondary antibody to provide a measurable signal.
When conjugated, a primary antibody allows for the direct detection of the target antigen of interest without needing a secondary antibody. Using direct detection has pros and cons, which are explored below.
Commonly used labels
Labels used to conjugate antibodies include fluorescent dyes, small molecules (eg, biotin), enzymes (horseradish peroxidase (HRP) or alkaline phosphatase (AP)), proteins (eg, R-PE, APC, streptavidin), latex or gold nanoparticles, and oligonucleotides. The choice of the label will depend on the experimental application, as shown in Table 1.
Table 1.Labels and their most common applications.
| Experimental technique | Label(s) |
| Western blot | HRP, AP, fluorescent dyes |
| Immunofluorescence | Fluorescent dyes, including Alexa Fluor®dyes |
| Fluorescent immunohistochemistry (singleplex) | Fluorescent dyes |
| Fluorescent immunohistochemistry (multiplex) | Fluorescent dyes, oligonucleotides |
| Chromogenic immunohistochemistry | HRP, AP, Biotin |
| Flow cytometry | Fluorescent dyes, such as Alexa Fluor®dyes, and fluorescent proteins, including Phycoerythrin (PE), Allophycocyanin (APC), and tandem dyes |
| ELISA, ELISA-based applications | HRP, Biotin |
| Immuno-PCR, Proximity ligation assay (PLA), Single-cell proteomics | Oligonucleotides |
| Lateral Flow Assay (LFA) | Latex and gold nanoparticles, Fluorescent nanoparticles (Europium) |
| Mass Cytometry (CyTOF®, Imaging Mass Cytometry™) | Metal ion tags |
How to conjugate antibodies?
Traditional covalent conjugation techniques, such as the NHS ester and two-tag methods, usually involveattaching the label to lysine residues. These conjugation protocols typically require knowledge of chemistry and experience in separation techniques, such as fixed-column chromatography, to remove low molecular weight activation reagents for the label and/or antibody.
Alternatively, simpler, three-step conjugation kits only require the addition of your antibody to a tube containing your desired label. These kits can help avoid common issues associated with the covalent conjugation process, including eliminating the need for separation steps to ensure the retention of your antibody.
For more information about quick & easy conjugation kits, read our page on Lightning-Link® kits.
Our comprehensive guide covers the ins and outs of antibody conjugation, including the difference between direct and indirect assays, common labels used, different chemistries behind antibody labeling, and methods you can use to conjugate your primary antibody.
If you’ve got any questions about our kits or conjugated antibodies, or you’d like to know about our custom conjugation solutions, we’d love to hear from you – get in touch.
Download our full guide to find out more about:
- Choosing between direct and indirect assays
- Commonly used labels
- Antibody labeling chemistries, such as the NHS ester method, two-tag method, and site-specific conjugation
- Conjugation kits
Alexa Fluor®is a registered trademark of Life Technologies. Alexa Fluor®dye conjugates contain(s) technology licensed to Abcam by Life Technologies.
FAQs
Antibody conjugation, also known as antibody labeling, is the process of linking an antibody to a specific tag or label. Most immunoassays require antibodies either directly conjugated to a specific label or detected by a conjugated secondary antibody to provide a measurable signal.
How much sodium azide to add to antibodies? ›
To prevent microbial contamination, you can add sodium azide to an antibody solution to a final concentration of 0.02% (w/v).
Are antibodies stable in PBS? ›
Store purified antibodies in PBS or Tris-Cl (50 mM, pH 8.0), unless there are reasons to do otherwise. Purified preparations of antibodies are stable in most commonly used buffers. Keep the pH near neutral (between 7 and 8).
What is conjugation strategy? ›
Conjugation Methods. Chemical conjugation methods are usually designed to modify the amine, carboxyl, or thiol groups of a protein (Figure 3). A variety of molecules have been conjugated to proteins, including nucleic acids, peptides, proteins, polymers, lipids, and NPs.
What is conjugation technique? ›
Conjugation is a gene transfer process in which a recipient bacterium receives DNA from a donor bacterium by cell-to-cell contact through conjugative pili. Conjugation is mediated by certain plasmids or transposons.
What is the purpose of adding sodium azide? ›
Sodium azide is used as a chemical preservative in hospitals and laboratories.
How to remove sodium azide from antibodies? ›
A Sephadex G25 column system or equivalent will effectively remove sodium azide from an antibody sample. Pre-packed Sephadex spin columns are readily available and can be used for this procedure.
How to prepare sodium azide solution? ›
To make a 10% stock solution of sodium azide, dissolve 10 g of sodium azide in 100 ml of distilled H2O. Store at room temperature.
Can you freeze HRP conjugated antibodies? ›
In contrast, enzyme-conjugated antibodies, such as HRP-conjugated antibodies, generally should not be frozen. Freezing and thawing these antibodies reduces the catalytic activity of the enzyme, leading to reduced signal when it is incubated with its substrate.
Why use HRP conjugated antibody? ›
HRP conjugates can be used for colorimetric detection. The conjugated reporter enzyme catalyzes the conversion of the chromogenic substrate to a colored precipitate, visualized directly on the blotting membrane or tissue sample.
Conjugation Protocol
Before adding the antibody to the Biotin (type A) Mix, add 1 µl of Modifier reagent to each 10 µl of antibody to be labelled. Mix gently. 2. Remove cap from vial of Biotin (type A) Mix and pipette the antibody sample (with added Modifier) directly onto the lyophilized material.
What happens if you freeze antibodies? ›
f. Repeated freezing and thawing kills antibodies. Once you have thawed an antibody solution, store it at 4°C for repeated use (unless you are aliquoting a newly arrived antibody; see next section).
At what temperature do antibodies denature? ›
IgG denaturation becomes significantly irreversible at temperatures higher than 65 °C (Mainer et al. 1999; Indyk et al. 2008). IgG almost completely loses its antigen-binding activity after heat treatment for several minutes at 90 °C (Augener and Grey 1970; van der Linden et al.
Do antibodies go bad? ›
Antibodies are relatively stable proteins and are resistant to a broad range of mild denaturing conditions. Most antibodies are stable for years when stored properly as per manufacturer's recommendations. In most cases antibodies can be stored at -20 °C without any loss in their binding capacity.
What is the purpose of antibody conjugation? ›
Antibody conjugation, also known as antibody labeling, is a technique for modification of antibodies which involves with the attachment of a specific tag to an antibody. These labeled antibodies can be used to isolate and purify a protein of interest from a complex mixture, usually cells, tissues or whole organisms.
What is DNA conjugation to antibodies? ›
DNA Origami or Hybridization Conjugation. AOCs with double-strand oligonucleotides could be obtained by a hybridization approach. A single strand oligonucleotide is first conjugated to an antibody and a complementary strand is hybridized to form a double-strand AOC.
What is antibody enzyme conjugate? ›
Conjugation of enzymes to antibodies involves the formation of a stable, covalent linkage between an enzyme [e.g., horseradish peroxidase (HRPO), urease, or alkaline phosphatase] and an antigen-specific monoclonal or polyclonal antibody in which neither the antigen-combining site of the antibody nor the active site of ...
What is conjugation of enzymes to antibodies? ›
Antibody-enzyme conjugates are generated by inducing covalent bonds between enzyme and antibody via chemical crosslinkers, enzyme catalysis or fusion proteins.
