Chromogenic Substrates Overview

Enzyme-Substrate Specificity

Enzymes are proteins that catalyze most of the chemical reactions that occur in the body, allowing these reactions to occur at neutral pH and body temperature. Enzymes are not modified or consumed during reactions, thus enabling their reuse in biochemical processes and their detection and study in the field of Clinical Microbiology and Biotechnology.

The chemical compound on which the enzyme exerts its catalytic activity is called a substrate. Each specific enzyme binds to its corresponding substrate and modifies it, promoting its transition from a reactive state to a product state. This substrate-enzyme interaction is fundamental for a large number of biological functions and metabolic processes. Enzymes are usually named according to the molecules they interact with – substrates – and their names typically end with the suffix “-ase”.


What are chromogenic substrates?

Chromogenic substrates are colourless soluble molecules consisting of a chromophore – a chemical group that, after enzymatic cleavage, releases colour – and a specific enzymatic substrate. They are synthetically produced and are designed to possess a selectivity similar to the natural substrate for the enzyme. These compounds are useful for enzymatic detection, as chromogenic substrates specifically bind with the target enzyme. This reaction allows the enzyme to catalyze the separation of the chromophore group, resulting in an insoluble product with a distinctive colour – releasing the chromophore – that confirms the existence and activity of the enzyme under study. This colour change can be followed spectrophotometrically and is proportional to the proteolytic activity of the enzyme.

Chromogenic substrates facilitate the quantitative and qualitative identification of enzymes and proteins in laboratory experiments, thanks to their visible colour change. This chromatic transition, whose intensity can be quantified, allows the precise measurement of the enzymatic or protein target in chromogenic assays. Its use is common in techniques such as Western blot, ELISA, immunohistochemistry, enzymatic assays, and microbial detection in culture media.

Common enzymes in chromogenic assays are Alkaline Phosphatase (AP), β-Galactosidase, and Horseradish Peroxidase (HRP).

DC Fine Chemicals, an international supplier dedicated to providing high-quality fine chemicals for production, that meet the needs of their customers, presents a new range of chromogenic products:

  • Lapis Substrates (Deep Blue)
  • X-Substrates (Blue Green)
  • Magenta Substrates (Magenta to Lilac)
  • Salmon Substrates (Pink)
  • Other Chromogenic Substrates


Clasification DCFC Code Substrates Synonims CAS Enzyme
Lapis Substrates

(Deep Blue)

125290 5-Bromo-3-indolyl phosphate disodium salt Blue-phos 16036-59-2 Alkaline phosphatase

(Blue Green)


125030 5-Bromo-4-chloro-3-indolyl-β-D-cellobioside X-Cellobioside 177966-52-8 β-Cellobiosidase
125000 5-Bromo-4-chloro-3-indolyl caprylate X-Caprylate 129541-42-0 Esterase
125220 5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside X-α-Gal, X-α-D-Galactoside 107021-38-5 α-Galactosidase
124980 5-Bromo-4-chloro-3-indolyl-α-D-glucopyranoside X-α-Glucoside 108789-36-2 α-Glucosidase
124960 5-Bromo-4-chloro-3-indolyl-N-acetyl-β-D-glucosaminide X-N-Acetyl-β-D-glucosaminide; X-Glucosaminide 4264-82-8 N-Acetyl-β-D-glucosaminidase
Magenta Substrates (Magenta to Lilac)


125020 5-Bromo-6-chloro-3-indolyl-β-D-glucopyranoside Magenta-glucoside 93863-89-9  β-Glucosidase
125010 5-Bromo-6-chloro-3-indolyl-α-D-glucopyranoside Magenta-α-D-glucoside 878495-64-8 α-Glucosidase
124990 5-Bromo-6-chloro-3-indolyl phosphate disodium salt Magenta-phos 404366-59-2 Alkaline phosphatase
124970 5-Bromo-6-chloro-3-indolyl-β-D-galactopyranoside Magenta-gal 93863-88-8 β-Galactosidase
125240 5-Bromo-6-chloro-3-indolyl-β-D-glucuronide, cyclohexylammonium salt Magenta-GlcA CHA salt, Magenta-gluc CHA salt 144110-43-0 β-Glucuronidase
Salmon Substrates



125260 6-Chloro-3-indolyl-α-D-galactopyranoside Salmon-α-gal 198402-61-8 α-Galactosidase
103380 6-Chloro-3-indolyl-β-D-galactopyranoside Salmon-gal 138182-21-5 β-Galactosidase
125250 6-Chloro-3-indolyl-β-D-glucopyranoside Salmon-glucoside 159954-28-6 β-Glucosidase
125230 6-Chloro-3-indolyl-β-D-glucuronide, cyclohexylammonium salt Salmon-glcA CHA salt, Salmon-gluc CHA salt 138182-20-4 β-Glucuronidase
Other Chromogenic Substrates

(Blackish purple)

125210 5-Bromo-4-chloro-3-indolyl phosphate disodium salt X-Phosphate disodium salt, BCIP 102185-33-1 Alkaline phosphatase, often in conjunction with NBT
125270 Nitro blue tetrazolium NBT, Nitro BT 298-83-9 Alkaline phosphatase

Use of BCIP-NBT substrates

The substrates Bromo-4-chloro-3-indolyl phosphate disodium (BCIP) and Nitro blue tetrazolium chloride (NBT) are commonly used together as a combination of chromogenic substrates. In experiments using BCIP-NBT, the corresponding enzyme linked to the probe antibody is alkaline phosphatase (AP). BCIP-NBT can be used in Western blot techniques, immunohistochemistry (IHC), and can be added to solid microbiological media to detect AP activity in microbial cultures.

The product formed is a dark purple colour that can be easily visible.

Figure 1: Chromogenic reaction using BCIP-NBT as substrate.

What are their applications?

Chromogenic substrates have a wide range of applications, mainly in:

  • Microbiology: They are used in culture media, clinical laboratories for diagnostic tests and identification of microorganisms, and in the food industry.
  • Biotechnology: Chromogenic substrates are vital in colourimetric detection. They are simple to use and suitable for a variety of immunotechniques, from immunohistochemistry to Western blot and ELISA.

Figure 2: Culture Media with chromogenic substrates from CHROMagar™  for detection of carbapenem-resistant Enterobacteria (CRE).


If you are interested in fine chemicals, be sure to visit our website, where you can also find chemical products that suit your business. If you need to know more about our chromogenic substrates or any other product, please contact us:



  1. Manafi, M. (29 November 1995). Fluorogenic and chromogenic enzyme substrates in culture media and identification tests. Hygiene Institute,University of Vienna.
  2. Druggan, P., & Iversen, C. (2014). Chromogenic Agars. In University of Dundee. Elsevier Ltd.
  3. Perry, J. D., & Freydière, A. M. The application of chromogenic media in clinical microbiology. Journal of Applied Microbiology.
  4. Jackson ImmunoResearch Laboratories,INC. Chromogenic Detection for Western Blot, IHC, and ELISA.


Article written by Sherry Cacay, Marketing Manager of DC Fine Chemicals


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