5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt
[CAS# 6578-06-9]

Identification

• Name: 5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt
• Synonyms: BCIP p-toluidine salt; X-phosphate p-toluidine salt
• Molecular Formula: C₈H₆BrClNO₄P.C₇H₉N
• Molecular Weight: 433.62
• CAS Registry Number: 6578-06-9
• EC Number: 229-506-1
• SMILES: CC1=CC=C(C=C1)N.C1=CC(=C(C2=C1NC=C2OP(=O)(O)O)Cl)Br

Properties

• Solubility: 1 mg/mL (water), 20 mg/mL (DMF)
• Melting point: 194-195 °C

Safety Data

• Hazard Symbols: GHS07 Warning
• Risk Statements: H302-H312-H315-H319-H332-H335
• Safety Statements: P261-P264-P264+P265-P270-P271-P280-P301+P317-P302+P352-P304+P340-P305+P351+P338-P317-P319-P321-P330-P332+P317-P337+P317-P362+P364-P403+P233-P405-P501

Hazard Classification

Hazard	Class	Category Code	Hazard Statement
Acute toxicity	Acute Tox.	4	H312
Acute toxicity	Acute Tox.	4	H332
Acute toxicity	Acute Tox.	4	H302
Eye irritation	Eye Irrit.	2	H319
Skin irritation	Skin Irrit.	2	H315
Specific target organ toxicity - single exposure	STOT SE	3	H335
Acute toxicity	Acute Tox.	3	H311
Carcinogenicity	Carc.	2	H351
Specific target organ toxicity - single exposure	STOT SE	2	H371
Acute toxicity	Acute Tox.	3	H301
Acute toxicity	Acute Tox.	3	H331
Skin sensitization	Skin Sens.	1	H317
Acute hazardous to the aquatic environment	Aquatic Acute	1	H400

Discovery and Applications

5-Bromo-4-chloro-3-indolylphosphate p-toluidine salt (BCIP) is a chromogenic substrate that was developed in the 1970s to improve molecular biology techniques. Researchers sought to create a stable and sensitive substrate for alkaline phosphatase, an enzyme commonly used in a variety of biochemical assays. BCIP became a key reagent in this quest due to its strong colorimetric properties, significantly improving the sensitivity and accuracy of enzyme detection methods.

The chemical formula of BCIP is C8H6BrClNO4P•C7H9N. It is a white to off-white powder that is soluble in organic solvents and slightly soluble in water. The molecule consists of a bromochloroindolylphosphate group paired with a p-toluidine salt, which stabilizes the compound and increases its solubility. After dephosphorylation by alkaline phosphatase, BCIP produces a deep blue color, making it very useful for visualization.

The chromogenic action of BCIP involves dephosphorylation by alkaline phosphatase. This enzymatic reaction removes the phosphate group from BCIP, forming an indigo dye. This color change is highly specific to the presence of alkaline phosphatase, making BCIP an excellent substrate for detecting and visualizing enzyme activity in a variety of assays.

BCIP is widely used in western blotting, where it serves as a substrate for alkaline phosphatase-conjugated antibodies. The enzyme-substrate reaction produces a blue precipitate at the site of the target protein, allowing researchers to detect and quantify specific proteins in a sample.

BCIP is used to stain tissues and cells in histochemical and immunohistochemical assays. The blue color produced by alkaline phosphatase activity contrasts sharply with the background, allowing the localization of specific enzymes, antigens, or other biomolecules in biological samples.

BCIP is used in in situ hybridization techniques to detect specific DNA or RNA sequences in tissue sections or cell preparations. When used with alkaline phosphatase-labeled probes, the substrate produces a blue signal that marks the location of the target nucleic acid.

BCIP is used in ELISA (enzyme-linked immunosorbent assay) assays to detect the presence of specific antigens or antibodies. The reaction of the substrate with alkaline phosphatase results in a color change that can be measured spectrophotometrically, providing quantitative data on the concentration of the analyte.

BCIP is safe when handled with standard laboratory precautions. It should be stored in a cool, dry place away from light to maintain its stability. When preparing BCIP solutions, it is recommended to wear appropriate personal protective equipment, including gloves and goggles, to avoid skin and eye contact. Adequate ventilation should be ensured to avoid inhalation of dust.

References

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2024. A versatile immunoassay based on functionalized nanoparticles for botulinum neurotoxin detection and sensor development. Discover Applied Sciences, 6(5).
DOI: 10.1007/s42452-024-05900-7