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Chromogenic Western Blotting

Chromogenic or precipitating substrates have been used widely for many years and offer the simplest and most cost-effective method of detection. When these substrates come in 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.

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Introduction

Unlike chemiluminscent or fluorescent blotting applications, chromogenic substrates do not require special equipment for visualization of the assay results. Similar to developing film, the blot is incubated in substate until the desired amount of development. Unlike chemiluminescent blotting assays, the colored precipitate can not be easily be stripped off reprobing. Therefore, it is imporant to allow the reaction to process until color development is satisfactory and then stop the reaction.

The low sensitivity of chromogenic substrates makes it difficult to optimize them for detecting proteins of low abundance. Though the reaction can be allowed to develop 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. 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. Since chromogenic substates are typically used to detect abundant proteins and reaction development can be monitored visually, this gives them greater flexibilty for optimization compared to chemiluminescent or fluorescent blotting systems.

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.

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Overview of Western Blotting

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Tech Tip #32: Guide to enzyme substrates for Western blotting

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Western Blotting Handbook

Chromogenic Horseradish Peroxidase Western Blot Substrates

Peroxide must be added to a substrate for colorimetric detection with 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 stablized peroxide in these solutions are generally concentrated and is less corrosive than the traditional 30% stock solution of hydrogen peroxide. Since 30% hydrogen peroxide and dilution solutions of hydrogen peroxide are not stable, reagents prepared with stabilized peroxide will provide more consistent results.

 
NBT/BCIP reaction diagram
Chromogenic substrates for Western blotting with HRP.

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. 1-Step TMB – Blotting 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 is well suited to applications that require a large signal-to-noise ratio.

4-CN 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.

DAB 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, CN/DAB Substrate. The CN/DAB Substrate has excellent sensitivity, yielding a dark black precipitate that photographs well. The CN/DAB Substrate works well in Western blotting and dot blotting applications.

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Chloronapthol

CN/DAB Substrate Kit

Precipitating TMB Substrate

Chromogenic Alkaline Phosphatase Western Blot Substrates

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.

BCIP has a molecular weight of 433.6, and hydrolysis by alkaline phosphatase results in a bluepurple 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.

Fast Red TR/AS-MX Substrate (structure pictured below) is a sensitive substrate that results in a bright red precipitate on transfer membranes. We no longer offer this particular substrate.

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NBT/BCIP Substrates

Filter Paper and Membranes

NBT/BCIP reaction diagram
NBT/BCIP reaction scheme. BCIP is hydrolyzed by Alkaline Phosphatase 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.
NBT/BCIP reaction diagram
Chromogenic substrates for Western blotting with Alkaline Phosphatase.
Written and/or reviewed by Douglas Hayworth, Ph.D.

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