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O Nitrophenyl Beta D Galactopyranoside

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The Curious Case of ONPG: Unlocking the Secrets of Beta-Galactosidase



Ever wondered how scientists track the activity of an enzyme, a microscopic workhorse driving countless biological processes? Imagine a tiny, cleverly designed molecular key that fits only a specific lock, revealing its activity through a dramatic color change. That's essentially what o-nitrophenyl β-D-galactopyranoside (ONPG) does. This seemingly simple molecule, often overlooked in the bustling world of biochemistry, plays a surprisingly crucial role in understanding and manipulating enzyme activity, particularly that of β-galactosidase. Let's delve into the fascinating world of ONPG and uncover its hidden potential.

Understanding the Molecular Key: Structure and Properties



ONPG is a synthetic substrate, meticulously crafted to interact with β-galactosidase. Its structure is a beautiful blend of two parts: o-nitrophenol (a colorful aromatic compound) and β-D-galactopyranoside (a sugar molecule mimicking lactose, the natural substrate of β-galactosidase). This clever design allows ONPG to act as a reporter molecule, subtly revealing the enzyme's presence and activity. The β-galactopyranoside portion fits snugly into the enzyme's active site, like a key in a lock. Once bound, β-galactosidase cleaves the molecule, releasing o-nitrophenol. This release is the key – o-nitrophenol, colorless in its galactoside form, turns a bright yellow in solution, providing a readily visible and quantifiable measure of enzyme activity. This color change is easily detected using a spectrophotometer, allowing for precise measurement of the reaction rate.

Applications in Research and Diagnostics: Beyond the Lab Bench



The simplicity and reliability of ONPG have cemented its place as a staple in various scientific disciplines. In microbiology, it's widely used to identify bacteria possessing β-galactosidase, crucial for lactose metabolism. A positive result – the appearance of yellow color – indicates the presence of this enzyme, aiding in bacterial identification and classification. This is particularly important in clinical diagnostics, helping to distinguish between different strains of bacteria based on their metabolic capabilities. For instance, differentiating E. coli strains based on their lactose fermentation capabilities often involves ONPG assays.

Furthermore, ONPG finds extensive use in molecular biology and biotechnology. It's a crucial tool in studying gene expression. By linking the expression of a gene to the production of β-galactosidase (often using reporter gene technology), scientists can quantify the gene's activity under different conditions. This is invaluable in understanding gene regulation, drug discovery, and the development of genetically modified organisms. For example, studying the effect of a particular drug on gene expression can be easily assessed by measuring the rate of ONPG hydrolysis in cells engineered to express the gene of interest.

Advantages and Limitations: A Balanced Perspective



ONPG offers several advantages. Its colorimetric assay is straightforward, requiring minimal equipment, making it accessible to a wide range of researchers. The reaction is relatively fast and readily quantifiable, providing reliable data. However, it's essential to acknowledge its limitations. ONPG is a synthetic substrate; its activity might not perfectly reflect the enzyme's activity with its natural substrate, lactose. The assay is also susceptible to interference from other compounds in the sample, potentially leading to inaccurate results if not properly controlled. Furthermore, its application is restricted to enzymes that specifically cleave β-galactosides.

Beyond the Basics: Exploring Advanced Applications



Recent advancements have explored ONPG's potential in new areas. For instance, researchers have incorporated ONPG into biosensors for detecting β-galactosidase activity in real-time. This opens doors for continuous monitoring of enzymatic processes in dynamic environments, such as in living cells or environmental samples. Moreover, derivatives of ONPG are being developed to improve sensitivity and specificity, addressing some of the limitations of the original compound.


Conclusion: A Versatile Tool with Enduring Relevance



o-Nitrophenyl β-D-galactopyranoside (ONPG) stands as a testament to the power of simple yet elegant tools in scientific research. Its unique ability to provide a readily quantifiable measure of β-galactosidase activity has revolutionized numerous fields, from basic microbiology to advanced biotechnology. Although it has limitations, its ease of use, cost-effectiveness, and versatility ensure that ONPG will continue to play a significant role in our understanding of enzymatic processes and their manipulation for years to come.


Expert-Level FAQs:



1. What are the potential sources of error in ONPG assays, and how can they be minimized? Errors can stem from improper sample preparation, interference from other chromophores in the sample, variations in temperature and pH, and enzyme instability. Minimizing these errors requires careful control of experimental conditions, appropriate sample preparation techniques (e.g., centrifugation, filtration), and the use of controls.

2. How can ONPG assays be adapted for high-throughput screening applications? ONPG assays are easily adaptable to high-throughput screening using automated plate readers capable of measuring absorbance at 420 nm. This allows for rapid screening of large libraries of compounds or mutants for their effects on β-galactosidase activity.

3. What are some alternative substrates for β-galactosidase, and how do they compare to ONPG? Alternatives include lactose, p-nitrophenyl β-D-galactopyranoside (PNPG), and fluorogenic substrates like 4-methylumbelliferyl β-D-galactopyranoside (MUG). While lactose is the natural substrate, it lacks the convenient colorimetric readout of ONPG. PNPG offers similar properties to ONPG, while MUG provides a more sensitive fluorometric assay. The choice depends on the specific application and desired sensitivity.

4. How can the kinetics of β-galactosidase be determined using ONPG? By measuring the rate of ONPG hydrolysis at various substrate concentrations, one can determine the Michaelis-Menten constant (Km) and the maximum reaction velocity (Vmax), providing valuable insights into the enzyme's catalytic efficiency.

5. Can ONPG be used to study β-galactosidase activity in vivo? While challenging, adaptations of the ONPG assay can be used to study β-galactosidase activity in vivo. This often involves microinjection of ONPG into tissues or cells followed by fluorescence or colorimetric detection. However, this approach requires careful consideration of factors such as substrate penetration and potential toxicity.

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o-Nitrophenyl-b-D-Galactopyranoside (ONPG) Test Principle, … 10 May 2024 · o-Nitrophenyl-b-D-Galactopyranoside (ONPG) Test Overview. In order for bacteria to ferment lactose, two enzymes, permease and beta-galactosidase, must be present. Lactose is transported into the bacterial cell wall by permease and subsequently hydrolyzed by beta-galactosidase into glucose and galactose. bacteria are able to digest glucose and ...

2-Nitrophenyl b- D -galactopyranoside = 98 enzymatic 369-07-3 2-Nitrophenyl β-D-galactopyranoside is an enzyme substrate used to detect lacZ activity and hence the presence of β-galactosidase.

ONPG Test (for β-galactosidase) - LaboratoryInfo.com 7 Mar 2022 · The test uses an artificial substrate called O-nitrophenyl-beta-D-galactopyranoside, which is similar in structure to that of lactose. The sugar is replaced with the o-nitrophenyl group, which has the ability to penetrate the cell in the absence of permease.

Chlorophenol Red-b- D -galactopyranoside = 90 HPLC 99792-79-7 A sensitive method has been developed for the detection of E. coli beta-galactosidase in transfected HeLa cells. The chromogenic substrate, CPRG (chlorophenol red-beta-D-galactopyranoside), was compared with ONPG (o-nitrophenyl-beta-D-galactopyranoside) by kinetic analysis with purified beta-galactosidase. The Km for CPRG was 1.35

19: ONPG Assay - Biology LibreTexts 21 Oct 2024 · O-nitrophenyl β-d-galactopyranoside (ONPG) is a synthetic compound that is similar in structure to lactose, the natural substrate of β-gal. β-gal can use ONPG as a substrate and will cleave ONPG to produce galactose and o-nitrophenol. O-nitrophenol has a yellow color and results in a yellow color change.

ortho-Nitrophenyl-β-galactoside - Wikipedia ortho-Nitrophenyl-β-galactoside (ONPG) is a colorimetric and spectrophotometric substrate for detection of β-galactosidase activity. [1] This compound is normally colorless. However, if β-galactosidase is present, it hydrolyzes the ONPG molecule into galactose and ortho-nitrophenol.

2-Nitrophenyl-beta-D-galactopyranoside | 369-07-3 - ChemicalBook 27 Jan 2025 · 2-Nitrophenyl-β-D-galactopyranoside (ONPG) is a colorimetric and spectrophotometric substrate for detecting β-galactosidase activity. This compound is usually colorless. However, if β-galactosidase is present, it hydrolyzes the ONPG molecule into galactose and ortho-nitrophenol.

ONPG test : Principle, Steps, Results and Interpretation O-nitrophenyl-beta-D-galactopyranoside (ONPG) is an artificial substrate structurally similar to lactose except that glucose is substituted with an o-nitrophenyl group. Unlike lactose, ONPG is able to enter the bacterial cell without the presence of permease.

2-Nitrophenyl- -D-galactopyranoside = 98 HPLC 369-07-3 2-Nitrophenyl-ß-D-galactopyranoside ≥98% (HPLC); CAS Number: 369-07-3; Synonyms: 2-Nitrophenyl-ß-D-galactopyranoside at Sigma-Aldrich

o -Nitrophenyl-b-D-galactopyranoside - MilliporeSigma o-Nitrophenyl-β-D-galactopyranoside, A β-galactosidase substrate for colorimetric and EIA applications; counterpart of widely used pNPP/alkaline phosphatase substrate. storage temp.

ONPG (β-galactosidase) Test: Principle, Procedure and Results On hydrolysis, through the action of the enzyme β-galactosidase, ONPG cleaves into two residues, galactose and o-nitrophenol. It is colorless compound: O-nitrophenol is yellow, providing visual evidence of hydrolysis.

O-Nitrophenyl-β-D-Galactopyranoside (ONPG) test: Principle, … 19 Jul 2020 · In this test, O-Nitrophenyl-β-D-galactopyranoside (ONPG) (artificial in nature) acts as a substrate for beta-galactosidase to ascertain the specific enzyme activity that serves in the identification and differentiation of organisms.

o-Nitrophenyl β-D-Galactopyranoside (CAS 369-07-3) o-Nitrophenyl β-D-Galactopyranoside: A chromogenic substrate for β-galactosidase. CAS Number: 369-07-3. Synonyms: ortho-Nitrophenyl β-D-galactopyranoside, o-Nitrophenyl β-D-Galactoside, ortho-Nitrophenyl β-D-Galactoside, NSC 83631, ONPG. Purity: ≥98%.

o-Nitrophenyl-β-D-galactopyranoside - NIST Chemistry WebBook o-Nitrophenyl-β-D-galactopyranoside. Formula: C 12 H 15 NO 8; Molecular weight: 301.2494

o -Nitrophenyl-b-D-galactopyranoside - MilliporeSigma o-Nitrophenyl-β-D-galactopyranoside ≥98% (HPLC), solid, β-galactosidase substrate, Calbiochem® CAS Number: 369-07-3; Synonyms: o-Nitrophenyl-β-D-galactopyranoside,o-NPG at Sigma-Aldrich

R Technical Data - himedialabs.com ONPG (Ortho-nitrophenyl beta-D-galactopyranoside) is a synthetic colourless compound (galactoside) structurally similar to lactose(1). β-galactosidase positive organisms cleaves ONPG to galactose and o-nitrophenyl, a yellow compound. This test is specially useful in the rapid identification of cryptic lactose fermenters (late fermenters).

o-Nitrophenyl beta-D-Galactopyranoside | CAS 369-07-3 - Biomol o-Nitrophenyl beta-D-galactopyranoside (ONPG) is a chromogenic substrate for beta-galactosidase. Upon enzymatic cleavage by beta-galactosidase, o-nitrophenol is released, which can be quantified by colorimetric detection at 420 nm as a measure of beta-galactosidase activity.Formal Name: 2-nitrophenyl beta-D-galactopyranoside. CAS Number: 369-07-3.

ONPG (o-nitrophenyl-β-D-galactopyranoside) - Thermo Fisher Scientific Thermo Scientific Pierce ONPG is the preferred colorimetric substrate for ELISA applications involving beta-galactosidase (b-Gal) as the reporter enzyme.

ONPG Test – Principle, Procedure, Uses and Interpretation 10 Aug 2022 · O-nitrophenyl-beta-D-galactopyranoside (ONPG), an artificial substrate, is incorporated into this test and acts as the substrate for the beta-galactosidase to ascertain the particular enzyme activity which subsequently aids in the identification and differentiation of different organisms.

Value of the O-nitrophenyl-beta-D-galactopyranoside test to ... A comparative study to determine beta-D-galactosidase activity among 171 strains of aerobic actinomycetes (including mycobacteria and rhodococci) was performed by using two growth media and four O-nitrophenyl-beta-D-galactopyranoside (ONPG) substrates.

ONPG Test- Principle, Procedure, Results, Uses - Microbe Notes 31 Mar 2023 · ONPG Test stands for O-Nitrophenyl-β-D-Galactopyranoside Test. The objective of ONPG is to determine the ability of an organism to produce β-galactosidase.

ONPG Test – ( o-nitrophenyl-β-D-galactoside) | Medical … ONPG is a rapid test that determine if an organisms ferment lactose by identifying the presence Beta-Galactosidase. This enzyme splits the B-Galactosidase bond in lactose, producingglucose and galactose.