Enzymes have been used as detection probes for years and are still commonly used, even with the upsurge of fluorescent probes, because of their simplicity, sensitivity and broad-spectrum applications. Enzyme probes, including horseradish peroxidase (HRP) and alkaline phosphatase (AP), are used to detect target proteins through chromogenic, chemiluminescent or fluorescent outputs. The variability of these readouts demonstrates the versatility that enzymatic probes have in biological research methods, including immunohistochemistry (IHC), immunoblotting and enzyme-linked immunosorbant assays (ELISAs).

Introduction to enzyme probes
Enzymes as detection probe reporters

Enzymes are used to detect target proteins by being conjugated to another protein, such as a primary or secondary antibody, biotin, avidin or some other protein that binds to the target protein. A substrate of the specific enzyme is then introduced, which interacts with the enzyme to form a visible reaction product that can be a dark precipitate or the emission of light or fluorescence. This signal is then detected through microscopic or scanning methods.

The benefits of using enzyme probes (also called reporters) to detect a target protein are three-fold:

  • High sensitivity – The signal output can be easily detected, and therefore low concentrations of target proteins can be identified. Additionally, methods of signal amplification are available that significantly increase the number of enzyme molecules to the site of the target protein. Finally, enzyme reporters exhibit rapid turnover, which increases the amount of substrate that a single enzyme converts during a given unit of time.
  • Long shelf life – The enzymes are quite stable when stored properly, and while the enzyme substrate is light-sensitive, the enzyme itself is not sensitive to degradation by ambient light.
  • Output versatility – Substrates that yield either chromogenic, chemiluminescent or fluorescent output are available for the most common enzyme probes.

Although these benefits demonstrate the versatility and convenience of enzyme probes, there are limitations to their use that should be considered when choosing the appropriate type of detection probe:

  • Large size – Enzyme reporters are considerably larger than organic fluorescent compounds (e.g., FITC, TRITC, AMCA) and therefore may interfere with the biological function of proteins to which they are conjugated.
  • Substrate requirement – Enzyme probes require the addition of a substrate for protein detection, and depending on the substrate used, this reaction can be sensitive to environmental conditions (e.g., light, temperature) and ambient light.
  • Endogenous interference – The enzymes used to detect target proteins in a sample are often expressed in the experimental system used (e.g., tissues, cells), which will also process the substrate and yield nonspecific background signal unless inhibited.

Learn more

Common enzyme probe reporters and chromogenic substrates

Enzymes function as assay reporters by reacting with substrates to produce colored, fluorescent or chemiluminescent signals that can be measured. Substrates can be either soluble or precipitating (see subsequent discussions below).

Enzyme Label Substrates
peroxidase (HRP)
3,3'-diaminobenzidine (DAB)
3,3',5,5'-tetramethylbenzidine (TMB)
2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS)
o-phenylenediamine dihydrochloride (OPD)
phosphatase (AP)
Combination of nitro blue tetrazolium chloride (NBT)
and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)
p-Nitrophenyl Phosphate (PNPP)
Glucose oxidase Nitro blue tetrazolium chloride (NBT)
β-galactosidase 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (BCIG or X-Gal)

Assay Development Technical Handbook

The revised Assay Development Technical Handbook is an essential resource for any laboratory using enzyme-linked immunosorbent assay (ELISA) and related plate-based assay methods. The handbook describes the essential techniques and tools for designing and optimizing ELISA Assays. Featured products include coated microplates, standards, blockers, buffers, probe-labeling reagents, secondary antibodies and detection substrates.

Contents include: Introduction to ELISA, Selecting an ELISA Plate, Thermo Scientific Pierce Microplates, Thermo Scientific Pierce Coated Microplates, Blocking and Washing, Blocking and Washing Reagents, Detection Probes, Antibody Labeling, Choosing a Substrate, Bulk and Custom Offerings, and Recommended Reading.

  Download the Assay Development Technical Handbook

Applications for enzyme probes

Enzyme probes are widely used in many different experimental applications because of their versatility, their ability to be conjugated to many different kinds of macromolecules and the availability and variability of substrates. Enzymes are most commonly used to detect proteins via direct and indirect antibody detection strategies, where the enzyme is conjugated to either the primary antibody that binds the target protein or the secondary antibody that targets the primary antibody, respectively. The enzyme is also often conjugated to biotin or avidin for use in avidin-biotin signal amplification systems.

Chromogenic detection using precipitating substrates is the most common type of protein targeting used for immunohistochemistry (IHC), although in recent years the use of fluorescent probes has increased with technological advances and the greater availability of fluorescence detection instrumentation. Chemiluminescent substrates are the standard for protein detection by Western blot analysis because of the high sensitivity and linear range of detection. Precipitating chromogenic substrates and, in recent years, fluorescent substrates are also available for this application. Chemiluminescent, fluorescent and soluble chromogenic ELISA substrates are commonly used, and each type of substrate exhibits characteristics specific for different experimental strategies.

Download documents

Enzyme reporters for probes
Horseradish peroxidase

Horseradish peroxidase (HRP) catalyzes the transfer of two electrons from a substrate to hydrogen peroxide to produce an oxidized substrate and water. For protein detection, HRP substrates (listed in the table below) are designed to generate a chromogenic, chemiluminescent or fluorescent signal upon oxidation. HRP has a molecular weight of 40,000, which is relatively small compared to other enzyme conjugates. This small size allows greater penetration into sample tissues and cells and reduces the likelihood of interfering with the conjugated protein function. HRP also has four lysines that are available for conjugation, which improves the efficiency of crosslinking to a protein of interest.

HRP has a high turnover rate and produces abundant reaction products in a short amount of time at physiological pH (7.6). HRP-IgG conjugates are superior to alkaline phosphatase and β-galactosidase conjugates due to their higher specific enzyme activity (more HRP/mole of antibody) and immunological reactivity (less steric hindrance because of the size of HRP).

One of the primary problems associated with the use of HRP is nonspecific staining that results from endogenous peroxide activity in certain tissues. Cryostat sections often contain a large amount of endogenous peroxidase activity, although commercial peroxidase inhibitors are available to reduce or eliminate endogenous peroxidase activity. HRP is the enzymatic label of choice for staining paraffin-embedded sections, a method that inhibits endogenous activity.

A second drawback to HRP is its sensitivity to degradation by microorganisms as well as the antibacterial agents used to fight them. Sodium azide is a potent inhibitor of HRP, but the enzyme can be stored in 0.01% thimerosal. HRP is also inhibited by cyanides, sulfides and azides. A final drawback of HRP is the mutagenicity or carcinogenicity of the reaction products of some horseradish peroxidase substrates. If any of these problems are a major concern, other enzymatic markers may be preferable.

Alkaline phosphatase

Alkaline phosphatases (AP) are a widely distributed family of enzymes that hydrolyze phosphates from nucleotides and proteins. Optimal enzymatic activity occurs at pH 9.0-9.6; these enzymes are activated by divalent cations and inhibited by cysteine, cyanides, arsenate, inorganic phosphate and divalent cation chelators, such as EDTA.

There are two forms of alkaline phosphatase in mammals; one form is distributed in a variety of tissues and the other is found only in the intestine. The two forms are affected differently by inhibitors and heat inactivators. The use of 1mM levamisole in the substrate buffer will inhibit endogenous tissue alkaline phosphatase activity. Intestinal alkaline phosphatase activity may be inhibited by treating the sections prior to staining with 20% acetic acid at 4°C for 15 seconds (or with 2.3% periodic acid for 5 minutes), followed by 0.02% potassium borohydride for 2 minutes.

Calf intestinal alkaline phosphatase conjugates are ideal in applications where high endogenous peroxidase levels contraindicate the use of HRP conjugates, such as with cryostat sections where peroxidase inhibitors are ineffective.

When used as a label, calf intestinal alkaline phosphatase (molecular weight 140,000) offers several distinct advantages over other enzymes. Because reaction rates remain linear, sensitivity can be improved by allowing the reaction to proceed for longer periods of time. The activity of calf intestinal alkaline phosphatase is not affected by exposure to antibacterial agents, such as sodium azide or thimerosal, so it may be stored for long periods of time in nonsterile environments. Because the endogenous activity of non-intestinal alkaline phosphatase can be inhibited by 1mM levamisole, antibodies labeled with this enzyme can be used as markers with many different tissues.

Glucose oxidase

Glucose oxidase is an enzyme isolated from Aspergillus niger that catalyzes the oxidation of β-D-glucose to produce hydrogen peroxide and gluconic acid. Glucose oxidase is a dimeric glycoprotein with a molecular weight of 160,000. Inhibitors of glucose oxidase include Ag+, Hg2+, Cu2+p-chloromercuribenzoate and phenylmercuric acetate.

Glucose oxidase is often the label of choice for samples with high endogenous peroxidase or phosphatase activity, as there is no endogenous glucose oxidase activity in mammalian tissues. It is important to choose a glucose oxidase with low catalase activity, though, because catalase destroys the hydrogen peroxide end product in the reaction.


β-galactosidase is an enzyme isolated from E. coli that is capable of hydrolyzing a variety of galactopyranoside derivatives, which produce both water-soluble and water-insoluble products. NaCl is an activator and Mg2+ is a cofactor of this enzyme, and the optimum pH for β-galactosidase is 7.0-7.5. For immunohistochemical staining, potential interference from endogenous enzyme can be overcome by embedding the sample with paraffin.

β-Galactosidase is sensitive and demonstrates no endogenous activity in mammalian cells, and therefore it is useful in applications where endogenous enzyme activity is a persistent problem. β-Galactosidase has been successfully coupled by a variety of crosslinkers to IgG fragments, whole immunoglobulins and insulin. One disadvantage of β-galactosidase is a lack of substrate variety.

Types of substrates
Chromogenic substrates

Chromogenic substrates have been widely used for many years and offer the simplest and most cost-effective method of detection. These substrates are divided into two groups based on the nature of the product of the enzyme-substrate reaction. When precipitating substrates come in contact with the appropriate enzyme, they are converted to insoluble products that precipitate onto the sample or membrane. Because of the insoluble nature of these products, precipitating substrates are commonly used for IHC and Western blotting. Conversely, soluble substrates form water-soluble colored products that dissolve into the test solution and are commonly used in ELISA assays. Besides the difference in the nature of their products, these two types of chromogenic substrates differ in the detection instrumentation used. Precipitating substrates require no more specialized equipment than a light microscope to detect the presence, intensity and localization of the insoluble product, while a microplate reader is used to measure the absorbance and therefore the amount of soluble product in solution. Chromogenic blotting substrates are available in a wide variety of specifications and formats, and the appropriate substrate choice depends on the enzyme label, desired sensitivity and the form of the signal or method of detection needed.

For either type of chromogenic substrate, protein detection is similar to developing film; the sample is incubated in substrate until the color development is satisfactory, after which the reaction is halted and/or the amount of product measured. This approach allows the user to control the resolution of the data. The low sensitivity of chromogenic substrates, though, makes optimization of detecting low-abundance proteins difficult; although the reaction can be developed for several hours or even overnight, this also allows background signal to develop as well. Where chromogenic substrates fail in terms of sensitivity, they are ideal for applications where protein abundance is high. The signal is stable because the product of the substrate reaction is stable when stored properly, and hence chromogenic substrates do not typically have issues with false negative results that can occur with chemiluminescent substrates in Western blots (e.g., ghost bands).

Chromogenic substrates are typically used to detect abundant proteins, and reaction development can be monitored visually; this allows greater flexibility for optimization compared to chemiluminescent or fluorescent blotting systems. The performance of a particular substrate may vary dramatically when obtained from different suppliers, though, because performance can be affected by the concentration and purity of the substrate and by other additives and buffer components that are a part of the formulation.

Precipitating chromogenic substrates for HRP and AP. Precipitating substrates are used for Western blot and immunohistochemistry detection.
Substrate Format Enzyme Features Relative Sensitivity Signal color
DAB Dry powder HRP Can be formulated Medium Brown
reagent kit
HRP 50X more sensitive than
DAB alone
Highest Brown to
BCIP Dry powder AP Can be formulated Medium Blue to
NBT Dry powder AP and
Non-carcinogenic Medium

Blue to

1-step NBT/BCIP Single step,
AP Low background for high
High Black to
1-step NBT/BCIP
+ suppressor
Single step,
AP Contains levamisole for
endogenous peroxidase
High Black to
Soluble chromogenic substrates for HRP and AP. Soluble substrate are used for ELISA and other plate-based assays.
Substrate Format Enzyme Features Relative Sensitivity Signal color
ABTS Tablets HRP Can be formulated Low Green
1-step ABTS Ready-to-use HRP 40X more sensitive than ABTS Medium Green
OPD Powder or
HRP Can be formulated High Yellow to
1-step TMB Ready-to-use HRP Up to 3.5X more sensitive than
High Blue
PNPP Powder or
AP Can be formulated High Yellow
1-step PNPP Ready-to-use AP No dissolving or diluting required High Yellow
Chemiluminescent substrates

Chemiluminescent substrates are popular because they offer several advantages over other detection methods, including a large linear response range that allows the detection and quantitation of a wide range of protein concentrations. Luminol is one of the most commonly used chemiluminescent reagents; luminol is oxidized by peroxide to form the excited-state 3-aminophthalate, which decays to a lower energy state by releasing photons of light at a wavelength of 425nm.

Chemiluminescent substrates differ from other substrates in that the light detected is a transient product of the reaction that is only present while the enzyme-substrate reaction is occurring. This is in contrast to chromogenic substrates that produce a stable, colored product. In a chemiluminescent reaction, the substrate is the limiting reagent in the reaction; as it is exhausted, light production decreases and eventually ceases. A well-optimized procedure using the proper enzyme conjugate dilutions will produce a stable output of light for several hours, allowing consistent and sensitive detection of proteins.

Chemiluminescent substrates are commonly used for the short-term, rapid detection of protein detection by Western blot analysis using x-ray film, phosphorimagers or CCD cameras.

Download documents

  • Tech Tip #67: Chemiluminescent Western blotting technical guide and protocols

Fluorescent substrates

While fluorophore-tagged antibodies and other molecules are more commonly used to detect target proteins, fluorescent substrates are also available for enzymatic detection. These substrates remain non-fluorescent or emit low fluorescence until metabolized by the enzyme probe, after which they emit intense fluorescence. These substrates offer greater sensitivity and the ability for rapid quantitation because of fluorescent microscopic imaging, fluorescent microplate readers and analytical software.

Enzyme labeling

Several methods have been developed and used routinely to prepare antibody or other protein conjugates with enzymes such as HRP and AP. All kinds of enzyme-conjugated secondary antibodies are commercially available, but specialized primary antibodies and other protein probes may have to be labeled by the researcher.

Glutaraldehyde is one of the simplest crosslinking reagents used for protein conjugation. It reacts with amine groups to create crosslinks by one of several routes. Under reducing conditions, the aldehydes on both ends of glutaraldehyde couple with amines to form secondary amine linkages. The reagent is highly efficient at protein conjugation but has a tendency to form various high-molecular weight polymers, making results difficult to reproduce.

Periodate-activation followed by reductive amination is an excellent strategy for conjugation of HRP and other glycoproteins. Treatment of the glycosylated enzyme with periodate results in oxidation of sugar cis-diol groups (especially sialic acid, which is common in glycoprotein polysaccharides), resulting in formation of aldehyde groups. These aldehyde groups will then efficiently react (in the presence of the mild reductant cyanoborohydride) with primary amines of an added antibody or other molecule. The result is stable conjugation by permanent amide bonds.

Finally, Sulfo-SMCC or other heterobifunctional crosslinkers can be used to conjugate enzyme molecules to antibody or other proteins in single- or two-step methods. The latter approach enables more precise control of the crosslinking result, because the enzyme can be activated and purified in one step and then conjugated to the target protein in a second step. This limits the directionality of crosslinking to one specific orientation (e.g., amines on the enzyme to sulfhydryl groups on the antibody) and the possibility of over polymerization.

  1. Sambrook J. and Russell D. W. (2001) Molecular cloning : A laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.
Visistat reference component