X-GAL (BCIG) is a widely used chromogenic β-galactosidase substrate. X-GAL is a colorless compound until cleaved by β-galactosidase, at which point X-GAL turns to an insoluble and detectable blue compound, making X-GAL particularly useful in techniques such as blue-white screening for cloning in bacteria. X-GAL can also be used for detection of β-galactosidase activity^[1][2].

1. Solution preparation^[1]
1.1 Preparation of stock solution
Solvent: DMF or DMSO.
Concentration: 20 mg/mL is recommended.
Storage: Store at -20°C or -80°C in dark after aliquoting. Avoid repeated freezing and thawing.
1.2 Preparation of working solution
Dilute X-gal to 40 μg/mL with 1 mL agar medium, and add 1 μL of IPTG (HY-15921) (24 mg/mL) and 1 μL of AMP (HY-A0181) (100 mg/mL) (optimized according to the experiment).
Note: The working solution should be prepared and used immediately. Keep it away from light.

2. Blue/white colony screening
2.1 After the DNA fragment is inserted into the pUC series vector (or other vectors with lacZ, Amp genes) and then transformed into lacZ-deficient cells, apply the above-mentioned X-gal, IPTG, and AMP plate culture medium.
2.2 Incubate at 37°C until blue-white colonies appear on the agar surface.
2.3 Cultivate the blue-white colonies further and identify them by double enzyme digestion.
2.4 Aspirate the dye working solution, wash with PBS 2-3 times, and observe under a microscope.

When the cells (HUVEC for example) were grown in dishes to 80% confluence, 50 μmol/L H₂O₂ were added in the medium for 12 h^[2].
Subsequently, cells were harvested with trypsin/EDTA and washed three times with PBS and further fixed with 4% formaldehyde for 10 min.
As control, cells without H₂O₂ treatment were prepared in the same way. Cells were stained with by X-gal at 37 °C.

The intestinal tissue was excised and cut longitudinally. After the intestinal content was rinsed off with phosphate buffer solution, the intestinal tissue was placed with the inner surface facing up on the slide^[2].
Next, a single blue colony containing β-gal (E. coli DH5α containing pUC18) was dilution in 100 μL PBS and then 10 μL were added in the tissue.
After incubation for 1 h, the tissues were washed with PBS solution.
As control, tissues without the blue colony were prepared in the same way.
All tissues were fixed with 4% formaldehyde for 10 min and were further stained by adding X-gal at 37 °C.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Sanchez-Ramos J, et al. The X-gal caution in neural transplantation studies. Cell Transplant. 2000 Sep-Oct;9(5):657-67.  [Content Brief]

  [2]. Li S, et al. 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.  [Content Brief]