X-GAL (5-bromo-4-chloro-3-indolyl-β-D-galactoside), consisting of galactose linked to a substituted indole, is a histochemical substrate for β-galactosidase. This substrate yields a blue precipitate upon hydrolysis, making it suitable for use in immunoblotting or immunocytochemical assays., and thus is widely used a chromogenic β-galactosidase substrate. β-galactosidase cleaves X-gal and produce an insoluble blue compound, which is detectable. X-gal is often used in molecular biology to test for the presence of an enzyme, β-galactosidase. It is also used to detect activity of this enzyme in histochemistry and bacteriology. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo dye as a result of enzyme-catalyzed hydrolysis.

Physicochemical Properties

Molecular Formula	C₁₄H₁₅BRCLNO₆
Molecular Weight	408.63
Exact Mass	406.977
CAS #	7240-90-6
PubChem CID	65181
Appearance	White to off-white solid powder
Density	1.9±0.0 g/cm3
Boiling Point	673.9±0.0 °C at 760 mmHg
Melting Point	230 °C
Flash Point	361.3±0.0 °C
Vapour Pressure	0.0±0.0 mmHg at 25°C
Index of Refraction	1.731
LogP	0.62
Hydrogen Bond Donor Count	5
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	3
Heavy Atom Count	23
Complexity	421
Defined Atom Stereocenter Count	5
SMILES	C1=CC(=C(C2=C1NC=C2O[C@H]3[C@@H]([C@H]([C@H]([C@H](O3)CO)O)O)O)Cl)Br
InChi Key	OPIFSICVWOWJMJ-AEOCFKNESA-N
InChi Code	InChI=1S/C14H15BrClNO6/c15-5-1-2-6-9(10(5)16)7(3-17-6)22-14-13(21)12(20)11(19)8(4-18)23-14/h1-3,8,11-14,17-21H,4H2/t8-,11+,12+,13-,14-/m1/s1
Chemical Name	(2S,3R,4S,5R,6R)-2-[(5-bromo-4-chloro-1H-indol-3-yl)oxy]-6-(hydroxymethyl)oxane-3,4,5-triol
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

References

[1]. The X-gal caution in neural transplantation studies. Cell Transplant. 2000 Sep-Oct;9(5):657-67. [2]. In-situ SERS readout strategy to improve the reliability of beta-galactosidase activity assay based on X-gal staining in shortening incubation times. Talanta. 2021 Nov 1;234:122689.

Additional Infomation

5-bromo-4-chloro-3-indolyl beta-D-galactoside is an indolyl carbohydrate that is the beta-D-galactoside of 3-hydroxy-1H-indole in which the indole moiety is substituted at positions 4 and 5 by chlorine and bromine, respectively. It is used to test for the presence of an enzyme, beta-galactosidase, which cleaved the glycosidic bond to give 5-bromo-4-chloro-3-hydroxy-1H-indole, which immediately dimerises to give an intensely blue product. It has a role as a chromogenic compound. It is an indolyl carbohydrate, a beta-D-galactoside, an organochlorine compound, an organobromine compound and a D-aldohexose derivative.

Solubility Data

Solubility (In Vitro)

DMF : 200 mg/mL (~489.44 mM) DMSO : ~100 mg/mL (~244.72 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.09 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.09 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.08 mg/mL (5.09 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: ≥ 2 mg/mL (4.89 mM) (saturation unknown) in 10% DMF 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 5: ≥ 2 mg/mL (4.89 mM) (saturation unknown) in 10% DMF 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 6: ≥ 2 mg/mL (4.89 mM) (saturation unknown) in 10% DMF 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.4472 mL	12.2360 mL	24.4720 mL
	5 mM	0.4894 mL	2.4472 mL	4.8944 mL
	10 mM	0.2447 mL	1.2236 mL	2.4472 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.