Blue‑Gal

Blue‑Gal (CAS 97753‑82‑7), systematically 5‑bromo‑3‑indolyl β‑D‑galactopyranoside, is a synthetic β‑galactosidase‑specific chromogenic substrate in which a 5‑bromo‑3‑indoxyl aglycone is linked via a β‑glycosidic bond to D‑galactose. When hydrolyzed by β‑galactosidase (EC 3.2.1.23, LacZ) in bacterial or recombinant systems, the indoxyl moiety is released and oxidizes to form an insoluble blue indigo‑type precipitate, enabling clear visual detection of enzyme activity. Blue‑Gal is primarily used in blue–white screening of recombinant clones, where lacZ⁺ colonies (intact LacZ) stain blue and lacZ⁻ (insert‑containing) colonies remain white. It is also applied in enzymatic assays, immunoblotting, and immunocytochemical detection of β‑galactosidase. Compared with X‑Gal, Blue‑Gal often produces a darker, more intense blue color, improving contrast and readability on agar plates and in histochemical preparations. The compound is soluble in organic solvents such as DMSO and DMF and is supplied as a high‑purity, research‑grade powder compatible with standard molecular‑biology and microbiological buffers.

Appearance

• White to almost white crystalline or lyophilized powder.
• Fine, free‑flowing texture suitable for precise weighing and dissolution in aqueous‑organic solvent mixtures.

Source / synthesis

• Synthetically produced by coupling 5‑bromo‑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., Sigma‑Aldrich, Cayman Chemical, AG Scientific, InalcoPharm, SRL Chemicals) as a high‑purity, chromogenic reagent.

Molecular weight and structure

• Molecular formula: C14H16BrNO6.
• Molecular weight: 374.18–374.2 g/mol.
• Structure: 5‑bromo‑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 and other β‑glycosidases under standard cloning‑medium conditions, making it suitable for cloning and phenotypic screening.

Biological activity

• Acts as a chromogenic reporter for β‑galactosidase‑mediated hydrolysis of β‑D‑galactosyl linkages in lactose analogs and glycosides.
• Enzymatic cleavage yields 5‑bromo‑3‑indoxyl, which oxidizes and dimerizes into an insoluble blue pigment, enabling sensitive detection in bacterial colonies, enzyme assays, and histochemical preparations.

Purity and microbial contamination

• Typically supplied at ≥98% purity as determined by HPLC or equivalent methods (e.g., >98.0% HPLC, ≥98% purity).
• Certificates of analysis from major suppliers indicate low residual solvents, acceptable heavy‑metal content, and non‑sterile, low‑microbial‑contamination status suitable for molecular‑biology and cloning‑grade use.

Identity and quality control

• Identity confirmed by molecular formula, molecular weight, NMR, and HPLC profiles consistent with 5‑bromo‑3‑indolyl β‑D‑galactopyranoside (Bluo‑Gal).
• Quality‑control checks include appearance, solubility, spectral characteristics, and performance in a defined β‑galactosidase (LacZ) activity assay on agar plates.

Shelf life and storage

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

Application

• Blue–white selection of recombinant bacterial clones (lacZ⁺ vs lacZ⁻) on agar plates in cloning systems.
• Chromogenic β‑galactosidase assays in solution or on solid media for enzyme‑kinetic studies and activity profiling.
• Use in immunoblotting, immunohistochemistry, and cell‑based assays for localization of β‑galactosidase expression.

Key characteristics

• High sensitivity and low background due to formation of an insoluble deep‑blue 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‑assay applications.

Citations

• TCI Chemicals – 5‑Bromo‑3‑indolyl‑β‑D‑galactopyranoside (Bluo‑Gal, CAS 97753‑82‑7, >98.0% HPLC): https://www.tcichemicals.com/IN/en/p/B3470
• Sigma‑Aldrich – 5‑Bromo‑3‑indolyl‑β‑D‑galactopyranoside (CAS 97753‑82‑7, ≥98%): https://www.sigmaaldrich.com/US/en/product/sigma/b4387
• Sigma‑Aldrich – Bluo‑Gal reagent for selection of recombinant bacterial clones: https://www.sigmaaldrich.com/IN/en/product/sigma/b2904
• abcam – Bluo‑Gal, chromogenic β‑galactosidase substrate (cloning and blue–white selection): https://www.abcam.com/en-us/products/biochemicals/bluo-gal-chromogenic-beta-galactosidase-substrate-ab146379
• Cayman Chemical – 5‑Bromo‑3‑indolyl‑β‑D‑galactopyranoside (CAS 97753‑82‑7, ≥98%): https://www.caymanchem.com/product/35299/5-bromo-3-indolyl-beta-d-galactopyranoside
• AG Scientific – Bluo‑Gal (X‑1016‑100MG) β‑galactosidase substrate for molecular biology: https://agscientific.com/products/biochemicals/substrates/galactosidase-substrates/bluo-gal-100-mg.html
• SRL Chemicals – 5‑Bromo‑3‑Indolyl‑β‑D‑Galactopyranoside (Bluo‑Gal, 99%): https://www.srlchem.com/product/details/25/27348/5-Bromo-3-Indolyl-B-D-Galactopyranoside-Bluo-Gal-for-molecular-biology-99
• InalcoPharm – 5‑Bromo‑3‑indolyl‑β‑D‑galactopyranoside (Bluo‑Gal) technical data sheet (formula, MW, appearance): https://www.inalcopharm.com/download_files/15_12215032680.pdf
• PMC – LacZ β‑galactosidase: structure and function (including X‑Gal and related indolyl‑β‑galactoside substrates): https://pmc.ncbi.nlm.nih.gov/articles/PMC3575911/
• X‑Gal Wikipedia overview (for Blue‑Gal–X‑Gal dye‑chemistry and cloning‑context reference): https://en.wikipedia.org/wiki/X-gal