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| Product Name | Beta-Galactosidase (β-Gal) Enzymatic Activity Assay Reagent Kit |
| Catalog No. | ETR-HMM-0066 |
| Description | A versatile colorimetric and fluorometric assay system for measuring beta-galactosidase enzyme activity using the chromogenic substrate ONPG (o-nitrophenyl-β-D-galactopyranoside) or the fluorogenic substrate 4-methylumbelliferyl-β-D-galactopyranoside (MUG). Beta-galactosidase cleaves the glycosidic bond of these substrates to release the colored or fluorescent aglycone. |
| Intended Use | Quantifying beta-galactosidase reporter gene activity in cell transfection studies, assessing senescence-associated beta-galactosidase activity as a cellular aging biomarker, and measuring endogenous beta-galactosidase activity in bacterial, yeast, and mammalian cell lysates. |
| Principle / Technology | Beta-galactosidase (EC 3.2.1.23) hydrolyzes the β-galactosidic bond in the synthetic substrate ONPG, releasing o-nitrophenol (yellow product, absorbance 420 nm, pKa 7.2) and D-galactose; the fluorogenic substrate MUG releases 4-methylumbelliferone upon cleavage (Ex/Em: 365/445 nm) for improved sensitivity 100–1000× over ONPG; cellular senescence-associated beta-galactosidase (SA-β-Gal) is measured at pH 6.0 where only the senescent lysosomal enzyme remains active. |
| Detection Method | Colorimetric absorbance at 420 nm (ONPG method) or fluorescence (Ex 365 nm / Em 445 nm) using a microplate reader |
| Sample Type | Mammalian cell lysates (transfected with lacZ reporter, aging cells, senescent cells), E. coli and yeast lysates, purified beta-galactosidase enzyme preparations |
| Performance Range / Specifications | ONPG assay range: 0.01–5 μg/mL purified beta-galactosidase; MUG assay range (fluorometric): 10–1,000× more sensitive than ONPG; incubation time: 15–60 minutes depending on activity level and substrate used |
| Sensitivity / LOD | ONPG method lower limit: approximately 0.1 mU/mL activity; MUG fluorometric method detects approximately 0.0001 mU/mL |
| Specificity | Both ONPG and MUG substrates are highly specific for beta-galactosidase; glucosidase, galactosidases with different stereospecificity, and other glycosidases show minimal cleavage of these β-galactoside substrates; the pH 6.0 SA-β-Gal assay protocol exploits the lysosomal pH optimum unique to senescent cells |
| Reaction Conditions / Protocol | For ONPG colorimetric method: lyse cells in lysis buffer; centrifuge; add 50 μL lysate to 200 μL Assay Mix (ONPG in sodium phosphate buffer pH 7.4 at 37°C); incubate 5–60 minutes at 37°C; stop with 50 μL Stop Solution (Na2CO3); read at 420 nm. For pH 6.0 senescence assay: fix cells with glutaraldehyde; add X-gal staining solution (pH 6.0) overnight at 37°C; observe blue nuclei indicative of SA-β-Gal positive cells |
| Components / Formulation | ONPG Substrate (lyophilized, 10 mg vial), MUG Substrate (lyophilized, 5 mg vial), Assay Buffer (sodium phosphate pH 7.4 for standard assay, pH 6.0 buffer for SA-β-Gal), Stop Solution (Na2CO3), β-Galactosidase Positive Control (E. coli beta-galactosidase, lyophilized), X-gal Solution (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside, for staining histochemical SA-β-Gal) |
| Storage Conditions | Lyophilized substrates at -20°C protected from light and moisture for 12 months; Stop Solution at room temperature; buffers at 2–8°C; ONPG stock at -20°C after reconstitution, stable 3 months |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 200 colorimetric assays; 500 fluorometric assays; X-gal staining for 20 coverslips |
| Product Form | Lyophilized substrates and enzyme control with separate buffer and staining components |
| Quality Control | Each lot tested against purified E. coli beta-galactosidase reference standard; ONPG Km and Vmax within specified ranges; fluorometric substrate signal enhancement ≥100× over ONPG method; X-gal staining validated with replicatively senescent IMR-90 and WI-38 cell lines |
| Key Features | Dual-substrate kit enables both high-sensitivity (MUG) and quantitative reporter gene (ONPG) measurements from a single kit; pH 6.0 SA-β-Gal protocol enables in situ cellular senescence detection; X-gal component provides histochemical visualization; widely cited in aging, cell biology, and reporter gene publications |
For research use only, not for clinical use.
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