Chromogenic Substrates for Western Blotting - USA (2023)

Chromogenic substrates or precipitants have been widely used for many years and offer the simplest and cheapest method for Western blot detection. When these substrates come into contact with the appropriate enzyme, they are converted into colored, insoluble products that precipitate on the membrane. The resulting band or spot of color does not require special equipment to process or visualize. Chromogenic staining substrates are available in a variety of specifications and formats. The choice of the appropriate substrate depends on the enzyme label, the desired sensitivity, and the shape of the signal or the required detection method.

Unlike chemiluminescence or fluorescence staining applications, detection with chromogenic substrates does not require special equipment to visualize assay results. Similar to developer film, the blot is incubated on the substrate until the desired amount of development is achieved. Unlike the chemiluminescent Western blot, the colored precipitate formed by the chromogenic substrates cannot be easily scraped off to facilitate reproach 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 complicates optimization for the detection of low abundance proteins. Although one can run the reaction for several hours or even overnight, this also allows the background signal to continue to develop. Where chromogenic substrates fail in terms of sensitivity, they are ideal for high protein abundance applications. Since the substrate reaction product is a colored precipitate, the signal is stable; Therefore, chromogenic substrates generally do not have a problem with the false negative results (ghost bands) that can occur with chemiluminescent substrates. The performance of a given substrate can vary dramatically when sourced from different vendors. This is because performance can be affected by the concentration and purity of the substrate and by other additives and buffering components that are part of the formulation.

4-Chloro-1-naphthol (4CN) has a molecular weight of 178.6 and can be used for the chromogenic detection of HRP by blotting and histochemistry. This precipitate is not as sensitive or stable as TMB and DAB, but the alcohol-soluble precipitate is easily photographed and has a distinctive blue-purple color that can be useful in dual-stain applications.

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

The individual benefits of 4-CN and DAB are often combined in a single substrate mix, Thermo Scientific Pierce CN/DAB Substrate. The CN/DAB substrate has excellent sensitivity and provides a dark black precipitate that is easy to photograph and is suitable for Western blot and dot blot applications.

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 membranes or nylon. BCIP can be used as a chromogenic substrate for immunoblotting and immunohistochemical studies.

An ideal system for AP blotting or staining applications is the combination of NBT and BCIP. Together they produce a deep purple-black precipitate that offers much higher sensitivity than either substrate alone. This reaction occurs at a constant rate, allowing precise control over its relative sensitivity. NBT/BCIP characteristically produces sharp band resolution with little membrane background staining.