5-Bromo-6-chloro-1H-indol-3-ol dihydrogen phosphate disodium salt is a chromogenic substrate frequently utilized in molecular biology and biochemical assays, particularly for the detection of enzymatic activity such as alkaline phosphatase. The compound is a derivative of indole and features substitutions at the 5 and 6 positions with bromine and chlorine atoms, respectively. Additionally, it carries a phosphate group at the 3-position of the indole ring, rendering it suitable as a substrate that undergoes enzymatic hydrolysis.
This compound is part of a broader class of indolyl phosphate esters designed to yield a visible color change upon enzymatic cleavage. Specifically, when acted upon by alkaline phosphatase, the phosphate group is removed, leading to the generation of an indoxyl intermediate. This intermediate subsequently dimerizes and oxidizes to form an intensely colored insoluble indigo dye. The visible blue precipitate that forms upon reaction serves as a straightforward and effective readout in various assays, including immunoblotting, histochemistry, and DNA probe detection.
The compound’s indole-based chromophore is central to its functionality, allowing it to act as a sensitive reporter molecule. The inclusion of both bromine and chlorine enhances electron-withdrawing effects, which can influence both the color properties of the dye product and its reactivity with the enzyme. The disodium salt form of the molecule increases its solubility in aqueous media, making it convenient for incorporation into assay buffers and reaction mixtures.
5-Bromo-6-chloro-1H-indol-3-ol dihydrogen phosphate disodium salt is typically provided as a dry powder and can be dissolved in suitable buffers shortly before use. In practical applications, this substrate is often found in kit formats for use in Western blotting and in situ hybridization. It is particularly valued in non-radioactive detection systems, where safety, convenience, and ease of interpretation are critical. Due to its sensitivity and the high signal-to-noise ratio provided by the insoluble blue precipitate, it enables visualization of low-abundance targets without the need for complex instrumentation.
The choice of this compound in diagnostic and research settings is often dictated by its compatibility with other detection reagents, its stability, and its capacity to yield consistent and reproducible results. The development of indolyl phosphate derivatives like this one has significantly advanced the field of molecular biology by enabling clear, visible detection methods that are safe, robust, and adaptable to multiple formats.
In storage, it is recommended to protect the compound from light and moisture to maintain its stability and reactivity. Typically, it should be stored at low temperatures, often -20°C, in tightly sealed containers. Preparation of working solutions should be done using sterile, filtered buffers, and solutions should be used promptly to prevent degradation or loss of reactivity.
This compound's reliable performance and visible signal generation have ensured its continued use in a wide range of applications. From academic research to clinical diagnostics, 5-bromo-6-chloro-1H-indol-3-ol dihydrogen phosphate disodium salt continues to serve as a critical tool in enzymology and molecular detection workflows.
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