Green‑Gal

Green‑Gal (CAS 207598‑26‑3), systematically N‑methyl‑3‑indolyl β‑D‑galactopyranoside, is a synthetic β‑galactosidase‑specific chromogenic substrate in which an N‑methyl‑3‑indolyl (N‑methyl‑indoxyl) aglycone is linked via a β‑glycosidic bond to D‑galactose. When hydrolyzed by β‑galactosidase (EC 3.2.1.23) in bacterial or recombinant systems, the indoxyl moiety is released and oxidizes to form a green‑ or fluorescent‑green, water‑insoluble dye, enabling clear visual or fluorescent detection of enzyme activity. The green coloration distinguishes Green‑Gal from the blue precipitate of X‑Gal or Bluo‑Gal, making it particularly useful for multiplex assays, double‑labeling experiments, or cases where blue‑colored backgrounds interfere. Green‑Gal is applied in recombinant‑DNA cloning for color‑based or fluorescent‑based selection of lacZ⁺ vs lacZ⁻ colonies, in enzyme‑activity assays, and in histochemical or cell‑based reporter‑gene studies. The compound is typically soluble in aqueous or mixed aqueous‑organic buffers and is supplied as a crystalline, high‑purity powder suitable for molecular‑biology and microbiological workflows.

Appearance

Off‑white to pale yellow crystalline or lyophilized powder.
When dissolved in aqueous buffer, yields a clear solution that becomes fluorescent‑green after enzymatic hydrolysis, although the dry form itself is non‑fluorescent.

Source / synthesis

Synthetically produced by coupling N‑methyl‑3‑indoxyl with β‑D‑galactose under glycosylation conditions to form the β‑D‑galactopyranoside derivative.
Commercially manufactured by specialty enzyme‑substrate and biochemical suppliers (e.g., US Biological, eChemist, ChemicalBook‑linked vendors) as a highly purified reagent.

Molecular weight and structure

Molecular formula: $C_{15} H_{19} NO_{6}$ .
Molecular weight: 309.31 g/mol.
Structure: N‑methyl‑1H‑indol‑3‑yl β‑D‑galactopyranoside, with a β‑glycosidic linkage between the indole C3‑O and the D‑galactose ring.

Sugar specificity

Specifically hydrolyzed by β‑galactosidases (LacZ, EC 3.2.1.23) and other β‑galactoside‑cleaving enzymes.
Shows minimal cross‑reactivity with α‑glycosidases under standard molecular‑biology and cloning‑medium conditions, making it suitable for selective detection of β‑galactosidase activity.

Biological activity

Acts as a chromogenic and/or fluorogenic reporter for β‑galactosidase‑mediated hydrolysis of β‑D‑galactosyl linkages in lactose analogs and glycosides.
Enzymatic cleavage yields N‑methyl‑3‑indoxyl, which oxidizes and dimerizes into a green‑to‑fluorescent‑green, water‑insoluble pigment, enabling sensitive detection in colonies, gels, and histochemical preparations.

Purity and microbial contamination

Typically supplied at ≥95–98% purity as indicated by vendor data sheets (e.g., “highly purified” or “HPLC‑assayed”).
Described as non‑sterile, research‑grade material with low microbial contamination status, suitable for use in microbiological and biochemical workflows under aseptic conditions.

Identity and quality control

Identity inferred from molecular formula, molecular weight, and spectral data (UV/Vis, NMR, HPLC) consistent with N‑methyl‑3‑indolyl β‑D‑galactopyranoside.
Quality‑control procedures include checks for appearance, solubility, chromatographic purity, and performance in a defined β‑galactosidase assay on agar plates or in solution.

Shelf life and storage

Shelf life is generally 12–24 months when stored as a dry powder at –20 °C in a tightly sealed, desiccated container, protected from moisture and light.
Stock solutions (often in DMSO or methanol) should be prepared immediately before use or stored frozen for short periods to minimize degradation and nonspecific oxidation of the indoxyl moiety.

Application

Color‑based or fluorescent‑based selection of recombinant bacterial clones (lacZ⁺ vs lacZ⁻) on agar plates where a green stain is preferred over blue.
Chromogenic or fluorogenic β‑galactosidase assays in solution, gels, or on solid media for enzyme‑kinetic and inhibitor‑screening studies.
Use in histochemical, immunocytochemical, and reporter‑gene expression systems requiring a green‑channel readout.

Key characteristics

Produces a green‑or‑fluorescent‑green insoluble dye upon β‑galactosidase hydrolysis, providing a spectrally distinct alternative to blue‑forming substrates.

Defined β‑D‑galactopyranoside linkage with an N‑methyl‑indolyl aglycone, giving high selectivity for β‑galactosidases over many other glycosidases.
Crystalline, stable powder with a known molecular formula and weight, suitable for quantitative cloning, enzyme‑assay, and multiplex‑detection applications.

Citations

US Biological – N‑Methyl‑3‑indolyl‑β‑D‑galactopyranoside monohydrate (Green‑Gal analog, CAS 207598‑26‑3, product 257744): https://cdn.usbio.net/molecular-biology/257744/n-methyl-3-indolyl-b-d-galactopyranoside-monohydrate
eChemist – N‑Methyl‑3‑indolyl‑β‑D‑galactopyranoside (CAS 207598‑26‑3) product page: https://www.echemi.com/produce/pr23061592145-n-methyl-3-indolyl-beta-d-galactopyranoside.html
ChemicalBook – N‑Methyl‑3‑indolyl‑β‑D‑galactopyranoside (CAS 207598‑26‑3) technical data: https://www.chemicalbook.com/ChemicalProductProperty_EN_CB6770763.htm
PMCID review on β‑galactosidase characterization and applications (for enzyme‑substrate‑context reference): https://pmc.ncbi.nlm.nih.gov/articles/PMC5429307/
Google Patent CA1337966C – Substrates for β‑galactosidase (including indolyl‑β‑galactoside derivatives): https://patents.google.com/patent/CA1337966C/en
X‑Gal and related chromogenic substrates overview (ScienceDirect X‑Gal topic): https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/x-gal
abcam – Magenta‑Gal and Blue‑Gal substrates (for dye‑color and cloning‑application context comparison): https://www.abcam.com/en-us/products/biochemicals/magenta-gal-chromogenic-beta-galactosidase-substrate-ab146381
Biotium – Red‑β‑D‑Gal (for general indolyl‑β‑galactoside‑dye chemistry reference): https://biotium.com/product/red-b-d-gal-5-bromo-6-chloro-3-indolyl-b-d-galactopyranoside/
General glycosidase‑substrate catalog (Green‑Gal and related N‑methyl‑indolyl‑β‑D‑galactosides overview): https://www.chemicalbook.com/ (search for “207598‑26‑3”)
β‑Galactosidase applications in food and diagnostics (PMC review context for enzyme‑applications): https://pmc.ncbi.nlm.nih.gov/articles/PMC10304135/

Product Name	Green-Gal
Chemical Name	N-Methyl-3-indolyl β-D-galactopyranoside
CAS Number	207598-26-3
Molecular Formula	C15H19NO6
Molecular Weight	309.32
Purity	98%
Product Code	G01040009
Packaging	Vacuum foil packaging

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Name	8HQ-Gal
Synonyms	8-Hydroxyquinolyl-β-D-galactopyranoside
CAS Number	110379-84-8
Molecular Formula	C15H17NO6
Molecular Weight	307.30 g/mol
Specifications & Purity	98% (Vacuum foil packaging)

Name	S-Gal
Synonyms	3-Cyclohexenoesculetin-β-D-galactopyranoside
CAS Number	182805-65-8
Molecular Formula	C19H22O9
Molecular Weight	394.38 g/mol
Specifications & Purity	98% (Vacuum foil packaging)

Name	X-GlcA CHX Salt Monohydrate
Synonyms	5-Bromo-4-chloro-3-indolyl-β-D-glucuronic acid cyclohexylammonium salt monohydrate
CAS Number	114162-0
Molecular Formula	C20H26BrClN2O8
Molecular Weight	538.0 g/mol
Specifications & Purity	≥95% (Vacuum foil packaging)

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