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].

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
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.
3. Cellular level detection^[2]
3.1 When cells are cultured to 80% confluence in culture dishes, add 50 μmol/L H₂O₂ to the medium and incubate for 12 h.
3.2 Harvest cells with trypsin/EDTA, wash three times with PBS, and fix with 4% formaldehyde for 10 min.
3.3 Prepare cells without H₂O₂ treatment in the same way as the control group.
3.4 Perform X-gal staining at 37 ℃.
4. Tissue-level detection^[2]
4.1 Excise intestinal tissues and cut them longitudinally. Rinse off intestinal contents with phosphate buffered saline, then place the intestinal tissues on glass slides with the inner surface facing upward.
4.2 Dilute a single blue colony containing β-gal (Escherichia coli DH5α carrying pUC18) in 100 μL PBS, and then add 10 μL of the dilution to the tissues.
4.3 After incubating for 1 hour, wash the tissues with PBS solution. For the control group, prepare tissues without blue colonies using the same method.
4.4 Fix all tissues with 4% formaldehyde for 10 minutes, and further stain with X-gal at 37 ℃.

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]