Magenta‑Gal

Magenta‑Gal (CAS 93863‑88‑8), systematically 5‑bromo‑6‑chloro‑3‑indolyl β‑D‑galactopyranoside, is a synthetic β‑galactosidase‑specific chromogenic substrate in which a 5‑bromo‑6‑chloro‑3‑indoxyl aglycone is linked via a β‑glycosidic bond to D‑galactose. When hydrolyzed by β‑galactosidase (EC 3.2.1.23), the indoxyl moiety is released and undergoes aerial oxidation to form an insoluble red–magenta indigo‑type dye, enabling clear visual detection of enzyme activity. This substrate is primarily used in recombinant‑DNA cloning systems (e.g., lac‑based blue–white selection) to distinguish β‑galactosidase‑positive (lacZ⁺) colonies from β‑galactosidase‑negative (lacZ⁻) recombinant colonies. The magenta color offers better contrast than traditional X‑Gal‑based blue colonies, reducing misreads in complex or high‑density plating. Magenta‑Gal is compatible with standard agar media and molecular biology buffers, and is supplied as a high‑purity powder for research‑use‑only or molecular biology‑grade applications.

Appearance

• Off‑white to pale yellow crystalline powder or lyophilized solid.
• Free‑flowing, fine‑particle form suitable for accurate weighing and dissolution in organic solvents or aqueous‑organic mixtures.

Source / synthesis

• Synthetically produced by coupling 5‑bromo‑6‑chloro‑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 as a research‑ or molecular‑biology‑grade reagent (e.g., abcam, Sigma‑Aldrich, GBIOSciences, GoldBio).

Molecular weight and structure

• Molecular formula: C14H15BrClNO6\text{C}_{14}\text{H}_{15}\text{BrClNO}_{6}C14H15BrClNO6.
• Molecular weight: 408.63 g/mol.
• Structure: 5‑bromo‑6‑chloro‑1H‑indol‑3‑yl β‑D‑galactopyranoside, with a β‑glycosidic linkage between the indole C3‑O and the D‑galactose ring in the (2S,3R,4S,5R,6R) configuration.

Sugar specificity

• Specifically hydrolyzed by β‑galactosidases (EC 3.2.1.23) and β‑galactoside‑cleaving enzymes.
• Shows minimal cross‑reactivity with other glycosidases under standard assay conditions, making it highly selective for β‑galactosidase activity.

Biological activity

• Acts as a chromogenic reporter for β‑galactosidase‑mediated hydrolysis of β‑D‑galactosyl linkages in lactose analogs and glycosides.
• Upon cleavage, the 5‑bromo‑6‑chloro‑3‑indoxyl aglycone is oxidized to an insoluble red–magenta pigment, enabling sensitive, low‑background detection of enzyme activity in bacterial colonies, enzyme assays, and histochemical applications.

Purity and microbial contamination

• Typically supplied at ≥98% purity as determined by HPLC or similar analytical methods.
• Certificates of analysis confirm low residual solvents, acceptable heavy‑metal content, and non‑sterile or low‑contamination status suitable for molecular biology and microbiological use.

Identity and quality control

• Identity confirmed by NMR, HPLC, MS, and elemental analysis; major suppliers provide batch‑specific certificates of analysis.
• Quality‑control checks usually include appearance, solubility, spectral characteristics, and assay performance in a defined β‑galactosidase activity system.

Shelf life and storage

• Shelf life is generally 12–24 months when stored as a dry powder at –20 °C, protected from moisture and light, often desiccated.
• Aqueous or stock solutions are typically prepared immediately before use or stored frozen for short periods to minimize degradation and nonspecific oxidation of the indoxyl moiety.

Application

• Blue–white‑style selection of recombinant bacterial clones (lacZ⁺ vs lacZ⁻) on agar plates, replacing X‑Gal with a magenta color readout.
• Chromogenic β‑galactosidase assays in solution or on solid media for enzyme‑kinetic studies and activity profiling.
• Use in histochemistry and cell‑based assays where spatial localization of β‑galactosidase activity is required.

Key characteristics

• High sensitivity and low background due to formation of a red–magenta, water‑insoluble dye upon β‑galactosidase hydrolysis.
• Defined β‑D‑galactopyranoside linkage providing selectivity for β‑galactosidases over other glycosidases.
• Crystalline, stable powder with a known molecular formula and weight, suitable for quantitative cloning and enzyme‑activity assays.

citations

• abcam – Magenta‑Gal, chromogenic β‑galactosidase substrate: https://www.abcam.com/en-us/products/biochemicals/magenta-gal-chromogenic-beta-galactosidase-substrate-ab146381
• Sigma‑Aldrich – Magenta‑Gal reagent for selection of recombinant bacterial clones: https://www.sigmaaldrich.com/IN/en/product/sigma/b8931
• GoldBio – Magenta‑Gal product page: https://www.goldbio.com/products/magenta-gal
• RPI Corporation – Magenta‑Gal (5‑bromo‑6‑chloro‑3‑indolyl‑β‑D‑galactopyranoside): https://www.rpicorp.com/products/biochemicals/other-enzymes/magenta-gal-2.5-g.html and https://www.rpicorp.com/products/biochemicals/other-enzymes/magenta-gal-1-g.html
• GBIOSciences – 5‑Bromo‑6‑chloro‑3‑indoxyl‑β‑D‑galactopyranoside (Magenta‑GAL) entry: https://www.gbiosciences.com/Bioassays/Enzyme-Substrates/5-Bromo-6-chloro-3-indoxyl-beta-D-galactopyranoside
• Bioglyco – Magenta‑Gal product information: https://www.bioglyco.com/magenta-gal-item-917.html
• Creative Enzymes – Magenta‑Gal product data sheet: https://www.creative-enzymes.com/downpdf.php?pid=6466
• MedChemExpress – β‑galactosidase‑substrate overview (Rose‑β‑D‑Gal / Magenta‑Gal‑type context): https://www.medchemexpress.com/search.html?q=%E0%B0%BF-galactosidase+substrate&ft=&fa=&fp=
• PubChem – 5‑Bromo‑6‑chloro‑3‑indolyl‑β‑D‑galactoside entry (CAS 93863‑88‑8): https://pubchem.ncbi.nlm.nih.gov/compound/5-Bromo-6-chloro-3-indolyl-beta-D-galactoside
• General enzyme‑substrate catalog (X‑ and Magenta‑type β‑galactosidase substrates): https://www.gbiosciences.com/Bioassays/Enzyme-Substrates/ (Magenta‑Gal and related dyes overview)