Chromogenic Western Blot Detection

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.

The most common substrates for colorimetric HRP western blots are- TMB, 4-CN and DAB.

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 Ultra 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.

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. 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

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.

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.

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.