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.

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  • introduction
  • Horseradish peroxidase chromogenic western blot substrates
  • Chromogenic alkaline phosphatase western blot substrates

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  • Chromogenic Western blot detection
  • Western blot antibodies
  • Chlornaphthol Substrate Spray
  • CN/OBD Substrate Kit
  • TMB 1-Step Transfer Substrate Solution
  • 1-Step NBT/BCIP Substrate Solution
  • alkaline phosphatase antibodies
  • Western blot signal enhancer


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.

Technical Guide for Protein Detection

This 84-page guide provides a detailed look at the final 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 that suits your experimental needs and the instruments at your disposal. Whether for rapid visualization or precise quantification, single probe detection or multiplexing, Thermo Fisher Scientific offers a variety of reagents and kits for Western blot detection and downstream analysis.

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(Video) Western Blotting

Know more

  • Western Transfer Overview
  • Manual of Western Blot
  • Tech Tip #32: Guide to Western Blotting Enzyme Substrates
  • Support Center for Protein Electrophoresis and Western Blotting

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  • Chromogenic Western blot detection
  • Western Blot Workflow Selection Guide
(Video) Western blot protocol video

Horseradish peroxidase chromogenic western blot substrates

The peroxide must be added to a substrate for colorimetric detection of horseradish peroxidase (HRP). Due to its extremely short shelf life at the desired concentration, hydrogen peroxide has traditionally been added to a buffer with the substrate just prior to use. As a result, these substrates typically have a pot life of a few hours. Many commercially available precipitating HRP substrates are supplied or prepared in a stable peroxidase substrate buffer. The stabilized peroxide in these solutions is generally concentrated and less corrosive than the conventional 30% hydrogen peroxide stock solution. Since 30% hydrogen peroxide and dilute hydrogen peroxide solutions are not stable, reagents prepared with stabilized peroxide give more consistent results.

Chromogenic Substrates for Western Blotting - USA (2)

Chromogenic substrates for western blotting with HRP.

3,3',5,5'-tetramethylbenzidine (TMB) with a molecular weight of 240.4 is most commonly used as a substrate for HRP in ELISA. However, in the presence of HRP and peroxide, a blue, water-soluble product is produced that can be precipitated on a membrane. Thermo Scientific 1-Step TMB-Blotting Substrate Solution is a one-component peroxidase substrate for immunohistochemistry and Western blotting. Product precipitation results in dark blue bands where the enzyme resides. The 1-Step TMB-Blotting Substrate Solution is suitable for applications that require a high signal-to-noise ratio. The most common substrates for colorimetric HRP are described below.

Chromogenic Substrates for Western Blotting - USA (3)

Western Blot Chromogener mit 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. Proteins were transferred to nitrocellulose membranes and the membrane blocked with 5% skim milk in TBS + 0.05% Thermo Scientific Tween 20. After blocking, the membrane was incubated with 0.5 µg/ml polyclonal antibody to heat shock protein-86. The membrane was washed and then incubated with 0.2 µg/ml HRP-conjugated goat anti-rabbit IgG and then washed again. The membrane was placed in 10 ml of water.Ultra TMB 1-Step Transfer Solutionand color development was stopped after 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 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.

Know more

  • Western Transfer Overview
  • Manual of Western Blot

select products

  • Chromogenic Western blot detection
  • Western Blot Workflow Selection Guide
(Video) Western Blot / Protein Immunoblot explained

Chromogenic alkaline phosphatase western blot substrates

Nitroblue 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 produces NBT formazan, a highly colored water-insoluble product. The substrate is widely used for immunochemical assays and techniques because the color produced by formazan is linear and stable over a wide dynamic range.

Chromogenic Substrates for Western Blotting - USA (4)

Chromogenic Western blot using NBT/BCIP substrate.Identical gels and nitrocellulose stains of IL-6 protein samples (0.1 to 2 µg per well) were run and detected with identical reagents and conditions without membrane treatment (top) or enhancer treatment (bottom). The final recognition was with1-Step NBT/BCIP Substrate Solution Thermo Scientific.

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.

Chromogenic Substrates for Western Blotting - USA (5)
(Video) In the Lab - Western blotting

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

Chromogenic Substrates for Western Blotting - USA (6)

Chromogenic substrates for western blotting with alkaline phosphatase.

Know more

  • Western Transfer Overview
  • Manual of Western Blot

select products

  • 1-Step NBT/BCIP Substrate Solution
  • Western blot signal enhancer

Literature recommendations

Gallagher, S., Winston (tank transfer systems), S.E., Fuller (tank transfer systems), S.A. and Hurrell (tank transfer systems; reversible protein staining), J.G. (2011). Immunoblotting and immunodetection.Current protocols in cell biology52:6.2:6.2.1–6.2.28.


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