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| Product Name | Beta-Galactosidase Assay Kit, ONPG Substrate, Colorimetric |
| Catalog No. | ETR-HMM-0088 |
| Description | Colorimetric beta-galactosidase assay kit using o-nitrophenyl-beta-D-galactopyranoside (ONPG) as a chromogenic substrate for quantitative measurement of beta-galactosidase (lacZ) activity in cell lysates, bacterial cultures, yeast, and tissue extracts. ONPG is hydrolyzed by beta-galactosidase to release galactose and o-nitrophenol (ONP), a yellow compound that absorbs strongly at 420 nm. The assay is widely used for: measuring beta-galactosidase expression as a reporter for gene expression studies; determining transfection and transduction efficiency in lacZ reporter systems; enzyme activity quantification in biochemical characterization; and senescence-associated beta-galactosidase (SA-beta-Gal) activity measurement at pH 6.0 in mammalian cells. The kit includes ONPG substrate, reaction buffer, stop buffer, and a detailed protocol with recommendations for both standard (pH 7.0-7.5) and senescence-associated (pH 6.0) assay conditions. |
| Intended Use | Quantitative measurement of beta-galactosidase activity for: lacZ reporter gene expression analysis in transfected/transduced mammalian cell lines; bacterial and yeast lacZ reporter assays; enzyme kinetic studies; senescence-associated beta-galactosidase (SA-beta-Gal) activity in cultured cells and tissue sections at pH 6.0; enzyme inhibitor screening and characterization; quality control of recombinant beta-galactosidase production. |
| Principle / Technology | o-Nitrophenyl-beta-D-galactopyranoside (ONPG) is a colorless synthetic substrate that is hydrolyzed by beta-galactosidase at the beta-1,4-galactosidic bond, releasing galactose and o-nitrophenol (ONP). ONP ionizes in alkaline conditions (added stop buffer) to the o-nitrophenolate anion, which has a strong yellow color with absorption maximum at 420 nm (molar extinction coefficient = 4,500 M^-1 cm^-1). The rate of ONP production is directly proportional to beta-galactosidase activity over a defined range. |
| Detection Method | Prepare cell lysate or enzyme sample; add sample to assay buffer containing ONPG (1-4 mg/mL final); incubate at 30-37 C (15-60 min, monitor for yellow color development); stop reaction by adding 1 M Na2CO3 (raises pH to >11, inactivates enzyme and ionizes ONP); measure absorbance at 420 nm; calculate beta-galactosidase units (1 unit = amount hydrolyzing 1 umol ONPG per minute under specified conditions). |
| Sample Type | Mammalian cell lysates (transfected with lacZ reporter), bacterial lysates (E. coli lacZ+ strains), yeast lysates, tissue homogenates, purified beta-galactosidase, cell culture supernatant (for secreted reporter). |
| Performance Range / Specifications | Detection range: 0.01-10 mU beta-galactosidase per well; linear range: 0-0.8 A420 units (corresponds to 0-178 uM ONP); assay duration: 15-60 min at 37 C; applicable to samples with beta-galactosidase activity from 0.1 mU/mL to 100 U/mL. |
| Sensitivity / LOD | Detection of as little as 0.01 mU (0.01 nmol ONP/min) beta-galactosidase per well; sensitivity in cell-based assays: approximately 1,000 cells expressing moderate levels of lacZ. |
| Specificity | ONPG is a specific substrate for beta-galactosidase (beta-D-galactoside galactohydrolase); no significant hydrolysis by beta-glucosidase, alpha-galactosidase, or other glycosidases. Endogenous lysosomal beta-galactosidase in mammalian cells is inactive at the pH 7.0-7.5 used for bacterial/yeast lacZ assays but active at pH 4.0-5.0. At pH 6.0, only senescence-associated beta-galactosidase (lysosomal origin) is detected in mammalian cells. |
| Reaction Conditions / Protocol | Standard assay (pH 7.3, 37 C): 15-60 min; senescence assay (pH 6.0, 37 C): 2-24 h (low activity); stop with Na2CO3 to pH ~11; ONP color stable for at least 2 h at RT after stopping. |
| Components / Formulation | ONPG Substrate (lyophilized, 50 mg per vial, 2 vials), Assay Buffer (5x, pH 7.3, 25 mL), SA-beta-Gal Buffer (5x, pH 6.0, 10 mL — for senescence assay), Stop Solution (1 M Na2CO3, 25 mL), ONP Standard (1 mM, 1 mL — for standard curve), Lysis Buffer (5x, 25 mL), Protocol. |
| Storage Conditions | ONPG at -20 C desiccated; buffers at RT; ONP standard at 2-8 C protected from light; reconstituted ONPG at -20 C (stable 3 months). |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 1 kit (sufficient for approximately 500 tests in 200 uL assay volume, 96-well format). |
| Product Form | Lyophilized ONPG; liquid buffers; ONP standard solution. |
| Quality Control | Each lot tested with purified E. coli beta-galactosidase standard: linearity R2 >0.99 over 0-5 U/mL; sensitivity: 0.01 mU detectable; substrate stability: >95% ONPG remaining after 3 months at -20 C; blank absorbance (no enzyme) <0.05 at 420 nm. |
| Key Features | ONPG chromogenic substrate for quantitative assays; compatible with lacZ reporter system; includes senescence-associated assay buffer (pH 6.0); lyophilized ONPG for stability; ONP standard included; adaptable to 96-well plates. |
| Purity | ONPG purity >=99% by HPLC; no free o-nitrophenol in substrate; all buffers prepared with ultrapure water. |
| Concentration | ONPG working concentration: 1-4 mg/mL (3.3-13.3 mM); ONP standard: 1 mM; detection range: 0-0.8 A420 (0-178 uM ONP). |
| Activity / Unit Definition | 1 beta-galactosidase unit = 1 umol ONPG hydrolyzed per minute at 37 C, pH 7.3; specific activity of E. coli beta-galactosidase: approximately 300-500 U/mg. |
| Molecular Weight | ONPG: 301.25 g/mol; ONP: 139.11 g/mol; o-nitrophenolate anion: 139.11 g/mol. |
| Source / Origin | ONPG: synthetic (chemical synthesis); buffers and stop solution: analytical grade chemicals; no animal-derived components. |
| pH Range / Optimal pH | Standard assay: pH 7.0-7.5 (optimal for E. coli lacZ); SA-beta-Gal assay: pH 6.0 (optimal for mammalian senescence-associated activity); stop solution raises pH to ~11 for ONP ionization. |
| Shipping Conditions | Ambient temperature for buffers; ONPG on cold pack. |
| Expiration Date / Stability | 12 months at recommended storage; reconstituted ONPG: 3 months at -20 C; stop solution: stable at RT for 24 months. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. |
| Compatibility | Compatible with mammalian cell lysis buffers (RIPA, NP-40, Triton X-100-based). EDTA (up to 5 mM) does not interfere. Reducing agents (DTT, beta-mercaptoethanol) at moderate concentrations (<10 mM) do not significantly affect the assay. PMSF and other protease inhibitors should be added to lysis buffer fresh. Avoid phosphate buffers for cell lysis (phosphate precipitates magnesium, a required beta-galactosidase cofactor). Compatible with clear 96-well plates; ONP absorption at 420 nm does not interfere with BCA or Bradford protein assays (performed on separate aliquots). |
| Recommended Buffer System | 5x Assay Buffer (pH 7.3): 250 mM sodium phosphate pH 7.3, 5 mM MgCl2, 250 mM beta-mercaptoethanol; 5x SA-beta-Gal Buffer (pH 6.0): 100 mM citric acid-sodium phosphate pH 6.0, 750 mM NaCl, 25 mM MgCl2; Stop: 1 M Na2CO3. |
| Application Notes / Precautions | Beta-mercaptoethanol in the assay buffer is essential for optimal bacterial beta-galactosidase activity (maintains reduced state of active-site cysteine). ONPG solution should be prepared fresh or stored at -20 C protected from light (it is light-sensitive and slowly auto-hydrolyzes). For mammalian cell assays, use a dedicated SA-beta-Gal buffer at pH 6.0 with longer incubation (2-24 h) to detect senescence activity; verify that the signal is truly SA-beta-Gal by confirming absence of activity in young proliferating cells at pH 6.0. Normalize beta-galactosidase activity to total protein concentration (Bradford or BCA assay) or cell number for accurate comparisons. |
| Batch-to-Batch Consistency | ONPG purity >=99% by HPLC; standard curve linearity R2 >0.99; sensitivity 0.01 mU detectable; blank <0.05 A420. |
For research use only, not for clinical use.
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