HRP antibody labeling protocol
August 25th, 2017
Directly label your primary antibody with HRP
Horseradish peroxidase (HRP) is a 44kDa 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 which 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 ELISAs, 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 protocols that are traditionally very time consuming. 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 and protein labeling kits allow the direct conjugation of your antibody, protein or peptide to HRP for use in any application. The kits only requires 30 seconds hands-on time and conjugates are ready to use after only 3 hours. Furthermore there is no need for extra any wash or separation steps post conjugation.
Benefits of using Lightning-Link® HRP
- Quick and easy – 30 seconds hands-on time
- Adjustable ratios of Ab to HRP, between 1:1 and 1:4
- No separation steps involved, recover 100% of your antibody or protein!
- Potentially more sensitive when compared to traditional methods – so less antibody required (see figure 1 below)
- Label from as little as 10µg – scale up to a gram or more!
Figure 1. Western blot data comparing 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 the inhibition of Azide on Lightning-Link® HRP conjugation
The below table shows the buffer considerations we recommend when using Lightning-Link® HRP antibody labeling kits.
If the antibody or other biomolecule that you want to label is either at an unsuitable concentration or the buffer contains substances likely to interfere with the conjugation reaction, we have a range of purification kits available that are compatible with our Lightning-Link® technology. Take a look at our flow diagram for more information.
The HRP antibody labeling protocol
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 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:
- Obtain your antibody – from a commercial supplier or generate your own in-house
- Add 1µl of modifier provided in the kit for each 10µl of your antibody
- Next add your antibody (with the modifier) to the vial containing the label
- Incubate for 3 hours
- Stop the reaction by adding the quencher
- Your conjugate is ready to use in any immunoassay!
Please note this is an overview of the protocol, precise volumes and concentrations can be found in the Lightning-Link® HRP protocol here.
Storing your HRP Conjugate
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 up to 2 years. Other storage conditions (e.g. 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.
Maintaining HRP conjugate performance
The performance of HRP conjugates diminishes over time; a number of factors contribute towards this including:
- Inactivation of HRP
- Instability of the bond which links HRP to the antibody
- Microbial attack
- Denaturation of the antibody
- Loss of performance accelerates with increasing temperature and with increasing dilution of the conjugates
To avoid loss of performance, we have developed our LifeXtendTM HRP conjugate stabiliser, 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.
Find out more about LifeXtend here.