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Learn how to directly label your primary antibody with HRP and maintain the HRP-antibody conjugate performance.
Horseradish peroxidase (HRP) is a 44 kDa glycoprotein with 6 lysine residues, which can be conjugated to antibodies and proteins for use in a variety of applications.
The enzyme label can be visualized through chromogenic reactions. For example, diaminobenzidine (DAB) in the presence of hydrogen peroxide (H202) is converted into a water-insoluble brown pigment. Other substrates that can be used to measure horseradish peroxidase activity include ABTS, TMB, and TMBUS.
HRP is a popular detection label used in research. Antibody-HRP conjugates are commonly used in ELISA, IHC, and western blotting. HRP can be conjugated to the primary antibody for direct detection or secondary antibody for indirect detection.
Direct detection is often a preferred method within these applications to avoid cross-species reactivity and to eliminate additional laborious wash and separation steps, especially in time-consuming protocols. However, directly conjugating HRP to the antibody or protein of choice can be difficult and labor-intensive if using traditional methodology.
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 only requires 30 seconds of hands-on time, and conjugates are ready to use after only 3 hours. Furthermore, there is no need for extra wash or separation steps post conjugation.
Figure 1. Western blot data comparing the working concentration of primary antibody conjugated to HRP using Lightning-Link® versus indirectly conjugated antibody. Note the working concentration of the directly labeled antibody is much lower than the indirectly labeled antibody whilst producing almost identical results, indicating greater sensitivity.
Before you begin
The composition of your antibody buffer is important when using Lightning-Link® HRP to conjugate your antibody or protein. Some substances such as BSA, gelatin, and Tris interfere with the labeling reaction. Figure 2 below shows the effect of buffer additives on both the Lightning-Link® chemistry and the enzyme activity.
In particular, it is important to note that sodium azide should not be present at all in the antibody buffer when conjugating to HRP as it significantly inhibits the conjugation reaction even in small concentrations (Figure 3).
Figure 2. The effects of additives on Lightning-Link® HRP conjugation.
Figure 3. Concentration curve showing that azide inhibits Lightning-Link® HRP conjugation.
The table below shows the buffer considerations we recommend when using Lightning-Link® HRP antibody labeling kits.
Amine free buffer (eg MES, MOPS, HEPES, PBS)
|Non-buffering salts (eg sodium chloride)||✓|
|Chelating agents (eg EDTA)||✓|
|Nucleophilic components (Primary amines, eg amino acids or ethanolamine, and thiols, eg mercaptoethanol or DTT)||✕|
1Please note that individual components used in the recommended concentrations should not affect the reaction. However, in combination with additional compounds that are not recommended above a certain concentration, the reaction may be affected.
2If you intend to use this kit for immunohistochemistry, we recommend avoiding gelatin or BSA.
One of the main benefits of using Lightning-Link® HRP to label your antibody or protein is that the conjugation process is quick and easy, requiring only 30 seconds of hands-on time and a 3-hour incubation before your HRP conjugate is ready to use in your application.
All you need to do is:
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. The best conditions for any particular conjugate must be determined by experimentation.
The performance of HRP conjugates diminishes over time; several factors contribute towards this including:
To avoid loss of performance, we have developed our LifeXtendTM HRP conjugate stabilizer, a proprietary multi-component reagent system that protects antibody-HRP conjugates from all of the negative factors noted above, thus ensuring the best possible performance in experiments performed at room temperature. Moreover, the ability to store HRP conjugates at working dilutions eliminates waste and improves consistency from experiment to experiment.