Chromogenic Western Blot Detection

Unlike chemiluminescent or fluorescent blotting applications, chromogenic substrates do not require special equipment for visualization of the assay results. We offer several chromogenic substrates for your different experimental needs.

Metal Enhanced DAB Substrate

Metal Enhanced DAB Substrate
Most sensitive
Limit of detection: 17 pg
Color: Brown/Black
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:1,000
2°: 1:5,000-1:50,000
Features: 2-component reagent kit

1-Step Ultra TMB Blotting

1-Step Ultra TMB Blotting
Blue precipitate
Limit of detection: 20 pg
Color: Dark blue
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:1,000
2°: 1:5,000-1:10,000
Features: Ready-to-use solution 

1-Step Chloronaphthol

1-Step Chloronaphthol
Blue-Purple precipitate
Limit of detection:
5 ng
Color: Blue-purple
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:500
2°: 1:2,000-1:20,000
Features: Raedy-to-use solution

CN/DAB Substrate kit

CN/DAB Substrate kit
Blue-Purple Precipitate
Limit of detection: 500 pg
Color: Black
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:500
2°: 1:2,000-1:20,000
Features: 2-component reagent kit

Membrane treatment for enhanced signal-to-noise

Thermo Fisher Scientific product Description Order
SuperSignal Western Blot Enhancer Membrane treatment reagent and a primary antibody diluent that increases both signal intensity and sensitivity 3- to 10-fold compared to detection performed without it. 46641

1-Step NBT/BCIP

1-Step NBT/BCIP
Ready to use solution
Limit of detection: 30 pg
Color: Black-purple
Recommend antibody dilutions (1 mg/ml stock):
1°: 1: 500
2°: 1:5,000-1:50,000
When to use: Antibodies or Probes conjugated to AP  

Additional products for AP substrates

Thermo Fisher Scientific product Description Order
WesternBreeze Chromogenic Kit, anti-mouse Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using mouse primary antibodies WB7103
WesternBreeze Chromogenic Kit, anti-rabbit Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using rabbit primary antibodies WB7105
WesternBreeze Chromogenic Kit, anti-goat Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using goat primary antibodies WB7107
SuperSignal Western Blot Enhancer Membrane treatment reagent and a primary antibody diluent that increases both signal intensity and sensitivity 3- to 10-fold compared to detection performed without it. 46641

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.

Chromogenic Western Blot Detection

Key Highlights

  • No special instrumentation required
  • Most cost-effective technique for high abundant targets

Chromogenic substrates for western blotting with horseradish peroxidase (HRP)

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

Chromogenic substrates

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.

Chromogenic alkaline phosphatase western blot substrates

Metal Enhanced DAB Substrate

Metal Enhanced DAB Substrate
Most sensitive
Limit of detection: 17 pg
Color: Brown/Black
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:1,000
2°: 1:5,000-1:50,000
Features: 2-component reagent kit

1-Step Ultra TMB Blotting

1-Step Ultra TMB Blotting
Blue precipitate
Limit of detection: 20 pg
Color: Dark blue
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:1,000
2°: 1:5,000-1:10,000
Features: Ready-to-use solution 

1-Step Chloronaphthol

1-Step Chloronaphthol
Blue-Purple precipitate
Limit of detection:
5 ng
Color: Blue-purple
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:500
2°: 1:2,000-1:20,000
Features: Raedy-to-use solution

CN/DAB Substrate kit

CN/DAB Substrate kit
Blue-Purple Precipitate
Limit of detection: 500 pg
Color: Black
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:500
2°: 1:2,000-1:20,000
Features: 2-component reagent kit

Membrane treatment for enhanced signal-to-noise

Thermo Fisher Scientific product Description Order
SuperSignal Western Blot Enhancer Membrane treatment reagent and a primary antibody diluent that increases both signal intensity and sensitivity 3- to 10-fold compared to detection performed without it. 46641

1-Step NBT/BCIP

1-Step NBT/BCIP
Ready to use solution
Limit of detection: 30 pg
Color: Black-purple
Recommend antibody dilutions (1 mg/ml stock):
1°: 1: 500
2°: 1:5,000-1:50,000
When to use: Antibodies or Probes conjugated to AP  

Additional products for AP substrates

Thermo Fisher Scientific product Description Order
WesternBreeze Chromogenic Kit, anti-mouse Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using mouse primary antibodies WB7103
WesternBreeze Chromogenic Kit, anti-rabbit Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using rabbit primary antibodies WB7105
WesternBreeze Chromogenic Kit, anti-goat Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using goat primary antibodies WB7107
SuperSignal Western Blot Enhancer Membrane treatment reagent and a primary antibody diluent that increases both signal intensity and sensitivity 3- to 10-fold compared to detection performed without it. 46641

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.

Chromogenic Western Blot Detection

Key Highlights

  • No special instrumentation required
  • Most cost-effective technique for high abundant targets

Chromogenic substrates for western blotting with horseradish peroxidase (HRP)

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

Chromogenic substrates

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.

Chromogenic alkaline phosphatase western blot substrates