Chromogenic or precipitating substrates have been used widely for many years and offer the simplest and most cost-effective method of western blot detection. When these substrates come into contact with the appropriate enzyme, they are converted to insoluble, colored products that precipitate onto the membrane. The resulting colored band or spot requires no special equipment for processing or visualizing. Chromogenic blotting substrates are available in a variety specifications and formats. The appropriate substrate choice depends on the enzyme label, desired sensitivity, and form of signal or method of detection needed.

Introduction

Unlike chemiluminescent or fluorescent blotting applications, detection with chromogenic substrates does not require special equipment for visualizing assay results. Similar to developing film, the blot is incubated in substrate until the desired amount of development is achieved. In contrast to chemiluminescent western blotting, the colored precipitate formed by chromogenic substrates cannot be easily stripped off to facilitate re-probing procedures. Therefore, it is important to allow the reaction to proceed until color development is satisfactory and then stop the reaction.

The low sensitivity of chromogenic substrates makes it difficult to optimize for detecting proteins of low abundance. Although the reaction can be allowed to develop for several hours or even overnight, this also allows background signal to continue to develop. Where chromogenic substrates fail in terms of sensitivity, they are ideal for applications where protein abundance is high. Because the product of the substrate reaction is a colored precipitate, the signal is stable; therefore, chromogenic substrates do not typically have issues with false negative results (ghost bands) that can occur with chemiluminescent substrates. The performance of a particular substrate may vary dramatically when obtained from different suppliers. This is 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.

Protein Detection Technical Handbook

This 84-page handbook provides a deep dive into the last step in the western blot workflow—protein detection. With a variety of detection techniques to choose from (chemiluminescence, fluorescence or chromogenic), you can select a technology to match your experimental requirements and the instruments you have available. Whether for quick visualization or precise quantitation, single-probe detection or multiplexing—Thermo Fisher Scientific offers a range of reagents and kits for western blot detection and subsequent analysis.

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Chromogenic horseradish peroxidase western blot substrates

Peroxide must be added to a substrate for colorimetric detection with horseradish peroxidase (HRP). Because of its extremely short shelf life at the desired concentration, hydrogen peroxide traditionally was added to a buffer, along with the substrate, immediately prior to use. As a result, these substrates typically have a useful shelf life of only a few hours. Many commercially available precipitating HRP substrates are supplied with, or come prepared in, stable peroxidase substrate buffer. The stabilized peroxide in these solutions is generally concentrated and less corrosive than the traditional 30% stock solution of hydrogen peroxide. Since 30% hydrogen peroxide and diluted solutions of hydrogen peroxide are not stable, reagents prepared with stabilized peroxide will provide more consistent results.

Chromogenic substrates for western blotting with HRP.

3,3′,5,5′-tetramethylbenzidine (TMB), with a molecular weight of 240.4, is most often used as a substrate for HRP in ELISAs. However, in the presence of HRP and peroxide, a water-soluble blue product is generated that can be precipitated onto a membrane. Thermo Scientific 1-Step TMB-Blotting Substrate Solution is a single-component peroxidase substrate for western blotting and immunohistochemistry. Precipitating the product results in dark blue bands where the enzyme is located. 1-Step TMB-Blotting Substrate Solution is well suited to applications that require a large signal-to-noise ratio. The most common substrates for colorimetric HRP are described below.

Chromogenic western blot using 1-Step Ultra TMB-Blotting Solution. Serial dilutions of HeLa cell lysate (7.5, 3.45, 1.88, 0.94, 0.47, 0.23, and 0.12 μg) were prepared and separated by electrophoresis. The proteins were transferred to nitrocellulose membranes and the membrane was blocked with 5% skim milk in TBS + 0.05% Thermo Scientific Tween 20. After blocking, the membrane was incubated with heat shock protein 86 polyclonal antibody at 0.5 μg/mL. The membrane was washed and then incubated with 0.2 μg/mL of HRP-conjugated goat anti-rabbit IgG and then washed again. The membrane was placed in 10 mL of 1-Step Ultra TMB-Blotting Solution and the color development was stopped at 5 minutes by rinsing the membrane with water.


4-chloro-1-naphthol (4CN) has a molecular weight of 178.6 and can be used for chromogenic detection of HRP in blotting and histochemistry. This precipitate is not as sensitive or as stable as TMB and DAB, but the alcohol-soluble precipitate photographs well and has a distinct blue-purple color that can be useful in double-staining applications.

Another widely used HRP substrate is 3, 3'-diaminobenzidine (DAB), which has a molecular weight of 214.1 and yields a brown precipitate in the presence of HRP and peroxide. The brown, insoluble product can be readily chelated with osmium tetroxide. This property makes DAB ideal for electron microscopy. The color produced by DAB can be intensified with the addition of metals such as nickel, copper, silver and cobalt that form complexes. The color produced by the metal complexes is darker than the color produced by DAB alone, enhancing the sensitivity in staining applications.

The individual benefits of 4-CN and DAB are often combined into a single substrate mixture, Thermo Scientific Pierce CN/DAB Substrate. The CN/DAB substrate has excellent sensitivity, yielding a dark black precipitate that photographs well, and it works well in western blotting and dot blotting applications.


Chromogenic alkaline phosphatase western blot substrates

Nitro blue tetrazolium (NBT), with a molecular weight of 817.6, is a member of a class of heterocyclic organic compounds known as tetrazolium salts. Upon reduction, the compound yields NBT-formazan, a highly colored, water-insoluble product. The substrate is widely used for immunochemical assays and techniques because the color produced by the formazan is linear and stable over a wide dynamic range.

Chromogenic western blot using NBT/BCIP substrate. Identical gels and nitrocellulose blots of IL-6 protein samples (0.1 to 2 µg per well) were probed and detected with identical reagents and conditions after no treatment (top) or enhancer treatment (bottom) of the membrane. Final detection was with Thermo Scientific 1-Step NBT/BCIP Substrate Solution.


5-bromo-4-chloro-3-indolyl phosphate (BCIP) has a molecular weight of 433.6, and hydrolysis by alkaline phosphatase (AP) results in a blue-purple precipitate that can be deposited on nitrocellulose or nylon membranes. BCIP can be used as a chromogenic substrate for both immunoblotting and immunohistochemical studies.

An ideal system for blotting or staining applications with AP is the combination of NBT and BCIP. Together, they yield an intense, black-purple precipitate that provides much greater sensitivity than either substrate alone. This reaction proceeds at a steady rate, allowing accurate control of its relative sensitivity. NBT/BCIP characteristically produces sharp band resolution with little background staining of the membrane.

NBT/BCIP reaction scheme. BCIP is hydrolyzed by AP to form an intermediate that undergoes dimerization to produce an indigo dye. The NBT is reduced to the NBT-formazan by the two reducing equivalents generated by the dimerization.

Chromogenic substrates for western blotting with alkaline phosphatase.


Recommended reading

Gallagher, S., Winston (tank transfer systems), S. E., Fuller (tank transfer systems), S. A. and Hurrell (tank transfer systems; reversible staining of proteins), J. G. (2011) Immunoblotting and immunodetection. Current Protocols in Cell Biology 52:6.2:6.2.1–6.2.28.