X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)

X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, CAS 7240-90-6) is the gold-standard chromogenic substrate for β-galactosidase (lacZ gene product) detection in molecular cloning, producing an insoluble intense blue precipitate (5,5′-dibromo-4,4′-dichloro-indigo) upon enzymatic hydrolysis that enables unambiguous blue/white colony screening of recombinant plasmids. With molecular formula C14H15BrClNO6 (MW 408.63 g/mol), this white crystalline compound features a β-D-galactopyranosyl moiety linked via O-glycosidic bond to the 3-position of 5-bromo-4-chloroindol-3-ol, remaining colorless and soluble until cleaved by E. coli β-galactosidase (α-complementation with lacZΔM15 hosts), liberating galactose and the indoxyl aglycone that auto-oxidizes/dimerizes to the highly colored, cell-localized indigo dye visible without equipment or washing. Essential for IPTG-inducible lacZ reporter systems, X-Gal revolutionized bacterial transformation workflows since 1982, supporting >1 million publications in gene expression analysis, promoter mapping, protein interaction studies (yeast two-hybrid), and phage display plaque purification. Incorporated into LB/X-Gal/IPTG plates at 40-100 μg/mL, it distinguishes recombinants (white, lacZ disrupted) from non-recombinants (blue, functional lacZ) with >99% accuracy, while fluorogenic alternatives (FDG/MUG) lack its archival stability and visual simplicity for routine lab use.

Appearance

• White to off-white crystalline powder.
• Hydrolysis yields insoluble intense blue precipitate.

Source

• Chemical synthesis: Koenigs-Knorr glycosylation of protected bromochloroindolyl alcohol + galactose.
• Commercial microbiology grade (Sigma-Aldrich >99%, Abcam >99%).

Molecular Weight and Structure

• Formula: C14H15BrClNO6; MW 408.63 g/mol.
• (2S,3R,4S,5R,6R)-2-(5-bromo-4-chloro-1H-indol-3-yloxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol.

Sugar Specificity

• Selective β-D-galactopyranoside for β-galactosidases (E. coli lacZ, mammalian lysosomal).
• Minimal cross-reactivity with glucosidases.

Biological Activity

• Chromogenic reporter; non-toxic to E. coli at working concentrations.
• 1000-fold color amplification localized to enzyme source.

Purity and Microbial Contamination

• 99% (HPLC); molecular biology grade.
• Synthetic; non-sterile reagent.

Identity and Quality Control

• [α]D -56° (c=1, DMF); mp 230°C (dec).
• Solubility 25 mM in DMSO/DMF; hydrolysis confirmed by blue product.

Shelf Life and Storage

• 5 years at -20°C desiccated; DMF/DMSO stock stable >1 year -20°C.
• Light/moisture protected.

Applications

• Blue/white screening; lacZ reporter gene assays.
• Yeast two-hybrid, phage titering, promoter analysis.

Key Characteristics

• Eye-readable; permanent blue stain; no-wash protocol.
• Gold standard since 1982; >10^6 citations.

Citation Links

• https://www.abcam.com/en-us/products/biochemicals/x-gal-chromogenic-beta-galactosidase-substrate-ab144388
• https://www.sigmaaldrich.com/US/en/product/sigma/b6024
• https://en.wikipedia.org/wiki/X-gal
• https://www.benchchem.com/pdf/X_Gal_A_Comprehensive_Technical_Guide_to_its_Mechanism_and_Application_in_Molecular_Biology.pdf
• https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/x-gal
• https://www.ibisci.com/products/x-gal
• https://www.biosynth.com/uploads/Brochures/Product%20Guides/Enzyme%20Substrates/X-Gal/X-Gal.pdf
• https://pubchem.ncbi.nlm.nih.gov/compound/65181 [ variant]
• https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/184/862/b6024pis.pdf
• https://www.glentham.com/en/products/product/GC2049/