Description
Product Overview
3,4-Cyclohexenoesculetin β-D-galactopyranoside is a chromogenic glycoside substrate designed for sensitive, selective detection of β-galactosidase activity. It serves as an autoclavable alternative to classic substrates (e.g. X-Gal), enabling robust blue/black coloration in bacterial colony screening and enzyme assays, especially in the presence of iron salts to form dark chelates.
As a reagent, it is particularly used in microbiology, molecular biology, and enzyme‐reporter systems (e.g. lac operon / lacZ studies) for distinguishing recombinant clones, screening β-galactosidase–expressing organisms, or probing enzyme kinetics.
Key Specifications
| Property |
Value / Description |
| Synonyms / Abbreviations |
CHE-Gal, S-Gal |
| CAS Number |
182805-65-8
|
| Molecular Formula |
C₁₉H₂₂O₉
|
| Molecular Weight |
~ 394.37 g/mol
|
| Appearance / Form |
Fine powder (white to beige) |
| Purity |
≥ 95 % (typical)
|
| Solubility |
Soluble in DMSO (~50 mg/mL)
|
| Storage / Handling |
Store in tightly closed container, desiccated, at room temperature (or per COA)
|
| Safety / Hazards |
Irritant to skin, eyes, respiratory tract (H315, H319, H335)
|
Functional Highlights & Advantages
-
Chromogenic reporter for β-galactosidase — upon enzymatic hydrolysis, the released aglycone (CHE) forms a deeply colored chelate (often black) with iron salts (Fe³⁺), providing high contrast and confined coloration on colonies or assay zones.
-
Autoclavable substrate — maintains activity or stability through sterilization, allowing incorporation into media or plates before autoclaving.
-
High localization — the dark precipitate remains strongly associated with the site of enzymatic reaction (i.e. the colony), improving resolution in mixed culture plates.
-
Broader organism compatibility — suitable for both Gram-negative and Gram-positive bacteria when used with metal ion chelation systems.
Suggested Applications & Usage Notes
-
Recombinant clone screening / Blue–black selection
Use CHE-Gal in agar or overlay media (with Fe³⁺) to detect β-galactosidase activity in lacZ+ clones. Positive clones hydrolyze the substrate and generate intense dark coloration, enabling visual discrimination.
-
Enzyme kinetics / β-galactosidase assays
In solution or plate-based assays, CHE-Gal may serve as a substrate to monitor enzymatic hydrolysis, especially where colorimetric readouts or precipitate formation is advantageous.
-
Mixed-culture plating
Because the colored product is tightly localized, CHE-Gal can reduce diffusion artifacts in dense or mixed bacterial populations, enhancing spatial resolution.
-
Media inclusion / pre-sterilization
Since CHE-Gal is autoclavable, it can be incorporated into growth media before sterilization, simplifying workflow and reducing contamination risk.
Suggested Handling & Protocol Tips
-
Always refer to the Certificate of Analysis (COA) for lot-specific details (e.g. actual purity, water content).
-
Prepare stocks in DMSO (or compatible solvent) at appropriate concentration; dilute into assay or media just prior to use.
-
Include Fe³⁺ salts (e.g. ferric ammonium citrate or other iron sources) in the assay to facilitate chelate formation and precipitate contrast.
-
Protect from moisture and light to preserve substrate integrity.
-
Validate working concentrations empirically (e.g. start with 100–500 µg/mL in plates or assay buffer) depending on enzyme expression levels.
-
When using in agar media, ensure even dispersion and pH compatibility; excess chelator or competing metal ions may reduce color yield.
