HRP antibody labeling with Lightning-Link®

Last edited Thu 26 June 2025

Horseradish peroxidase (HRP) is a 44 kDa glycoprotein with 6 lysine residues, which can be conjugated to antibody molecules and proteins for use in various applications. In the conjugation process, HRP is covalently attached to antibody molecules to facilitate detection and quantification in immunoassays. Antibody-HRP conjugates are commonly used in ELISA, IHC, western blotting, co-immunoprecipitation (co-IP), and biosensor development (al).

The enzyme label can be visualized through chromogenic reactions. For example, diaminobenzidine (DAB) in the presence of hydrogen peroxide (H0) is converted into a water-insoluble brown pigment. Other substrates measuring horseradish peroxidase activity include ABTS, TMB, and TMBUS. The presence of the target antigen is detected through the enzymatic reaction catalyzed by HRP, where the transfer of electrons during substrate oxidation generates a measurable signal. As shown in Fig 1, the data demonstrate the specificity of the assay, with representative images illustrating the detected signal.

HRP can be conjugated to the primary antibody for direct detection or the secondary antibody for indirect detection. These applications often prefer direct detection to avoid cross-species reactivity and eliminate additional laborious wash and separation steps, especially in time-consuming protocols. However, directly conjugating HRP to the antibody or protein of interest can be difficult and labor-intensive if using traditional methodology. It is important to include appropriate control samples in immunoassays to validate results and distinguish specific signals from background noise.

Our Lightning-Link® HRP antibody labeling kit allows the direct conjugation of your antibody, protein, or peptide to HRP for use in any application. The kit does not require additional steps to purify the antibody prior to conjugation, simplifying the workflow. The protocol allows you to mix your antibody with the lyophilized reagent, and an optional overnight incubation can be performed to improve results. Certain buffers should be chosen carefully to avoid components that may react with HRP conjugates, such as primary amines and thiols. Performing specific steps prior to others in the conjugation protocol maximizes efficiency and prevents undesired reactions. If using photo-crosslinking protocols, a light source (e.g., 365nm) is essential for site-specific antibody conjugation. There is significant interest in optimizing reaction conditions to improve conjugation efficiency and antibody performance.

Benefits of using Lightning-Link® HRP:

• Quick and easy – 30 seconds of hands-on time, ready to use after 3 hours of incubation

• Rapid production of high-quality conjugates, enabling efficient workflows

• Adjustable ratios of antibody to HRP, between 1:1 and 1:4

• No separation steps or extra wash steps are involved. Recover 100% of your antibody or protein

• Potentially more sensitive when compared to traditional methods – so less antibody required

• Label from as little as 10 µg – scale up to a gram or more

The kit is shipped at ambient temperature and should be checked upon receipt and stored according to the instructions to maintain product integrity. For technical support, to request a quote, or for more information, please contact us or visit our website.

Introduction to antibody labeling

Antibody labeling is a fundamental technique in scientific research, enabling precise detection and quantification of specific proteins or antigens within complex biological samples. This process involves covalently attaching a detectable marker, such as horseradish peroxidase (HRP), to an antibody molecule. Once labeled, the antibody can bind to its target antigen, allowing researchers to visualize or measure the presence of that antigen using a variety of methods, including ELISA, western blotting, and immunohistochemistry (IHC). The use of the HRP enzyme as a label is especially popular due to its high sensitivity and straightforward detection through colorimetric reactions. By directly labeling primary antibodies with HRP, researchers can streamline their protocols, minimize the need for secondary antibodies, and reduce the risk of cross-reactivity, ultimately improving the specificity and reliability of their results in applications ranging from protein detection to quantification in research settings.

Benefits of using the HRP enzyme

The HRP enzyme offers several key benefits when used for antibody labeling. Its high sensitivity allows for the detection of even low concentrations of antigens, making it ideal for applications where signal strength is critical. HRP catalyzes the oxidation of hydrogen peroxide, producing a colored product that is easily measured, which enhances the clarity and quantification of results. The enzyme’s activity is highly specific and can be finely tuned with the right substrates and buffers, ensuring reliable detection. Additionally, HRP’s relatively small size means it can penetrate tissues and cells more effectively, without hindering the function of the conjugated antibody or protein. By enabling direct conjugation to primary antibodies, HRP labeling reduces the reliance on secondary antibodies, which not only streamlines workflows but also increases the specificity of antigen detection. These benefits make HRP a preferred choice for researchers seeking robust, sensitive, and specific detection in a wide range of immunoassays.

Stage 1 - Choosing or preparing your antibody buffer

• Buffer preparation should be completed prior to starting the conjugation process.
• The composition of your antibody buffer is important when using Lightning-Link® HRP to conjugate your antibody or protein. Common buffer additives can hamper the conjugation process. Certain buffer components, such as primary amines and thiols, may react with HRP and should be avoided to ensure optimal conjugation results.
• When preparing the antibody buffer, thoroughly mix the antibody solution to ensure homogeneity before proceeding.

Steps

Ensure your antibody buffer is free from BSA, gelatine, and Tris as much as possible.

• We recommend reducing the presence of these additives to the concentrations below. However, if you intend to use this kit for immunohistochemistry, we recommend avoiding gelatin or BSA. Buffers can still contain amine-free buffer (eg MES, MOPS, HEPES, PBS), non-buffering salts (eg sodium chloride), chelating agents (eg EDTA), sugars or borate buffer.

Stage 2 - Procedure for HRP antibody conjugation

Steps

Obtain your antibody from a commercial supplier or generate it in-house

Add 1 µl of modifier provided in the kit for each 10 µl of your antibody.

Incubate for 3 hours.

Stop the reaction by adding the quencher.

Your conjugate is now ready to use in any immunoassay.

Stage 3 - Storing and maintaining performance

Steps

HRP antibody conjugates must be stored correctly to maintain their performance.

• The best conditions for any particular conjugate must be determined by experimentation.
• Initial storage at 4⁰C is recommended for 12–18 months (as long as your antibody can be stored at 4°C – check the manufacturer’s recommendation).
• A preservative may be desirable for long-term storage for up to 2 years. Other storage conditions (eg frozen at -70⁰C or stored at -20⁰C with 50% glycerol) may also be satisfactory.

However even with correct storage, the performance of HRP conjugates diminishes over time.

Loss of performance accelerates with increasing temperature and dilution of the conjugates. Several factors contribute to this including:

• Inactivation of HRP
• Instability of the bond, which links HRP to the antibody
• Microbial attack
• Denaturation of the antibody

Troubleshooting common issues

While modern conjugation kits have streamlined the antibody labeling process, challenges can still arise. Common issues include low conjugation efficiency, which can lead to weak detection signals, and instability of the conjugated antibody, resulting in diminished enzyme activity over time. To address these problems, researchers can adjust the ratio of antibody to HRP, modify reaction times, or optimize storage conditions to preserve enzyme activity and specificity. Ensuring the purity and quality of both the antibody and HRP enzyme is also critical for successful conjugation. If difficulties persist, consulting the technical support provided by the kit manufacturer or seeking advice from experienced colleagues can be invaluable. By understanding the key factors that influence antibody labeling and leveraging available resources, researchers can overcome common obstacles and achieve reliable detection and quantification of target antigens in their experiments.