Cell Wall-Bound Acid Invertase Activity Assay Kit (DNS, Micro Method)

Cat. No.: C1521860

GRADE & PURITY BioReagent

Panoramica

Sucrose invertase (Invertase, Ivr) irreversibly hydrolyzes sucrose into fructose and glucose and is one of the key enzymes in sucrose metabolism in higher plants. Based on its optimal pH, Ivr in higher plants is classified into acid invertase (AI) and neutral invertase (NI). The optimal pH for AI ranges from 3 to 5, and it is further divided into two forms: soluble AI (S-AI) and cell wall-bound insoluble AI (B-AI). B-AI is localized in the intercellular space and bound to the cell wall. Its primary function is to participate in the breakdown of sucrose during apoplastic unloading in the phloem, thereby maintaining the sucrose concentration gradient between source and sink tissues. In this assay kit, B-AI catalyzes the degradation of sucrose to produce reducing sugars, which then react with 3,5-dinitrosalicylic acid to form a reddish-brown amino compound. This compound exhibits a characteristic absorption peak at 510 nm. Within a certain range, the rate of increase in absorbance at 510 nm is proportional to B-AI activity.

Specifications

Specifiche e purezza

BioReagent

Grado

BioReagent

Applicazione

Enzyme activity assay

Tipo di campione

Tessuti vegetali

Metodo di rilevamento

Colorimetric Method

Detection instrument

Microplate reader,Visible Spectrophotometer

Detection wavelength

510nm

Stoccaggio e spedizione

Condizioni di conservazione di stoccaggio

Store at 2-8°C,Protected from light

Spedito in

Wet ice

Stabilità e conservazione

Store at 2-8℃ for 6 months. Store in the dark.

Application Protocols

Per instructions, only product-provided protocols should be listed here.

• Detailed step-by-step procedures, reaction volumes, incubation times, wavelengths, and calculation templates: Not specified for this item; refer to the kit Instruction Manual/CoA.

General recommendation

• Follow the kit insert precisely for sample preparation, assay setup, standard curve preparation, incubation, color development, and data analysis. Deviations from the validated procedure can alter linear range and accuracy.

Biological Roles

Cell wall-bound acid invertase (CWI) is a key enzyme in plant carbohydrate partitioning.

• Function (biology; literature): CWI (apoplastic β-fructofuranosidase) hydrolyzes sucrose into glucose and fructose in the cell wall/apoplast. This reaction drives phloem unloading by maintaining a sucrose gradient, supports sink strength, and provides hexoses for metabolism and cell wall biosynthesis.
• Developmental roles: High CWI activity correlates with active growth zones (young leaves, roots, developing seeds/fruits), seed filling, and tissue differentiation. It participates in cell expansion by supplying osmolytes and substrates for cellulose/hemicellulose synthesis.
• Regulation: CWI is regulated transcriptionally and post-translationally (e.g., by invertase inhibitors and wall association). Environmental cues (light, sugars, hormones such as auxin, ABA, cytokinin) modulate activity.
• Stress responses: Under drought, salinity, cold, and pathogen attack, CWI activity often changes, affecting osmotic adjustment and defense-associated sugar signaling. Altered CWI can influence source–sink balance and pathogen nutrition interfaces.
• Isoforms and localization: Distinct genes encode cell wall-bound (acid) vs soluble (vacuolar, acid) and cytosolic (neutral/alkaline) invertases. Proper fractionation is necessary to ascribe activity to the wall-bound pool.

This kit’s readout (DNS micro method) provides a quantitative measure of reducing sugars generated by CWI activity in cell wall-enriched preparations, enabling comparative studies across tissues, treatments, and genotypes in research settings. For research use only (from Product Data).

Buffer Applications

This product is not a buffer reagent per se. However, buffer choice is crucial for accurate measurement of cell wall-bound acid invertase activity.

General/literature guidance (not item-specific)

• Recommended pH window: Acid invertases exhibit maximal activity in pH ~4.5–5.5. Sodium acetate (50–200 mM) or citrate–phosphate buffers are commonly used.
• Ionic strength: Moderate ionic strength can stabilize enzyme conformation; excessive salt may alter wall association or interfere with extraction.
• Additives: PVPP (0.5–2% w/v) can be used during extraction to bind phenolics; avoid strong reductants (e.g., DTT, β-mercaptoethanol) in the assay step due to potential interference with DNS detection.
• Substrate solutions: Prepare sucrose fresh in assay buffer; heat gently to dissolve, cool, and equilibrate pH. Include substrate blanks.
• Temperature control: Maintain constant temperature during incubation (e.g., 30–37°C) using a thermostatted block or water bath; pH of weak acids shifts slightly with temperature—pre-equilibrate buffers accordingly.

For detailed buffer recipes and any kit-supplied buffers, Not specified for this item; refer to CoA/Spec Sheet.

Green Alternatives

DNS-based assays are robust but use nitroaromatic chemistry and boiling steps. Depending on your goals, greener or safer alternatives may be appropriate.

Comparison (literature; general guidance)

• DNS (this kit’s method)
  • Pros: High tolerance to many matrix components; well-established; inexpensive; broad linear range with proper calibration; compatible with micro-scale (microplates/cuvettes).
  • Cons: Uses nitroaromatic reagent in alkaline medium and requires heating; generates hazardous chemical waste; potential interference from strong reductants and phenolics.
• GOPOD (glucose oxidase–peroxidase) assay
  • Pros: Enzymatic, milder conditions, often room temperature to 37°C; fewer hazardous components; high specificity for glucose.
  • Cons: Measures glucose only (fructose requires isomerization or separate assay); susceptible to inhibitors in crude extracts; generally costlier.
• HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection)
  • Pros: Highly specific quantitation of glucose/fructose/sucrose without color reagents; multiplexing sugars.
  • Cons: Requires specialized instrumentation; higher energy/solvent footprint; not a rapid screen.
• Resorcinol- or pH-indicator-based methods
  • Pros: Simpler chemistry, less hazardous than DNS in some formulations.
  • Cons: Narrower applicability, lower robustness with complex matrices.

Sustainability tips

• Miniaturize volumes to reduce waste (consistent with “micro method” design).
• Consolidate heating steps and use sealed secondary containment to minimize exposure.
• Segregate DNS-containing waste for proper disposal; neutralize alkali after analysis where permitted by regulations.
• Consider enzymatic GOPOD confirmation for critical samples to reduce repeat DNS runs.

Pharmaceutical Uses

Not applicable. This product is an enzyme activity assay kit intended for basic and applied research in plant and related biological systems. It is not an excipient, processing aid, or analytical reagent defined by a pharmacopeial monograph in this context.

• Research Use Only (from Product Data): Not for diagnostic, therapeutic, or clinical use.
• If sugar analysis is required in regulated environments, validated compendial or ICH-aligned analytical methods should be selected. DNS-based reducing sugar methods exist in analytical literature but may not satisfy specificity requirements for QC of pharmaceutical substances without additional validation.

Physical Properties

This is a multi-reagent assay kit; standard single-compound physical constants (bp, mp, density, pKa, logP) do not apply.

Item-specific properties

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Kit component concentrations, pH, and volumes: Not specified for this item; refer to CoA/Spec Sheet.
• Storage conditions (from Product Data): Store at 2–8°C, protected from light. Shipped on wet ice.

General/literature context (for orientation only; not item specifications)

• DNS-based colorimetric assays develop an orange–red chromophore measured typically at 540 nm after heating with alkaline DNS reagent in the presence of reducing sugars. Exact absorbance maxima and extinction coefficients depend on formulation and matrix.
• Enzymatic reaction for acid invertase is typically performed in mildly acidic buffers (literature: pH ~4.5–5.5) and moderate temperatures (25–37°C), followed by detection.

Notes

• Because the kit comprises multiple liquids/solids, properties like refractive index, viscosity, and solubility are component-specific and must be confirmed from the kit’s Certificate of Analysis or Instruction Manual.
• Do not extrapolate generic DNS reagent properties to this specific product; consult the kit insert for validated handling and measurement parameters.

Quality and Grades

• Supplied grade: BioReagent (from Product Data). BioReagent grade typically signifies reagents and kit components suitable for biochemical/enzymatic assays, with attention to background absorbance, reagent stability, and impurity levels that could interfere with enzyme activity or colorimetric detection.

What BioReagent grade implies (general context)

• Low background in the relevant assay wavelength window to preserve sensitivity and linearity in micro-scale measurements.
• Component-to-component consistency across lots to support reproducibility of standard curves and activity determinations.
• Stabilization appropriate for lab research conditions (e.g., buffers adjusted for shelf-life at 2–8°C and protection from light). Specific stabilizers, if present, are Not specified for this item; refer to CoA/Spec Sheet.

Kit-specific quality considerations

• Enzyme activity assays depend critically on accurate standards. Verify the lot-specific calibration curve parameters and any provided reference standards in the kit insert (Not specified for this item; refer to CoA/Spec Sheet).
• Spectral performance: For DNS-based detection, low UV/Vis baseline turbidity and minimal dye impurities are important to achieve precise A540 readings in micro volumes.

Compliance and documentation

• For research use only (from Product Data). Not intended for diagnostic or clinical use.
• CoA/Spec Sheet will detail component identities, fill volumes, acceptance criteria, and recommended performance tests (e.g., linear range, LOD/LOQ) where applicable.

Reaction and Applications

Purpose and principle

• The kit quantifies the activity of cell wall-bound acid invertase (CWI; apoplastic β-fructofuranosidase), which hydrolyzes sucrose to glucose and fructose. The DNS micro method detects the reducing sugars formed by measuring the increase in absorbance of a reduced nitroaromatic chromophore at visible wavelengths (literature: ~540 nm), proportional to sugar concentration.

Applications (research use)

• Plant physiology: Assessing sink strength, phloem unloading capacity, and developmental regulation by measuring CWI in cell wall fractions from leaves, roots, fruits, and storage organs.
• Stress biology: Monitoring CWI modulation under abiotic (drought, salinity, cold) or biotic (pathogen interaction) conditions.
• Bioprocessing/agri-biotech: Screening germplasm or treatments that alter sucrose partitioning and cell wall metabolism.
• Method validation: Comparing CWI activity with soluble acid invertase (vacuolar) or neutral/alkaline invertases to dissect compartment-specific sucrose turnover.

Practical notes (literature, not item-specific)

• Extraction: CWI is ionically or covalently associated with the cell wall; pre-extraction with salt buffers (e.g., 0.5–1 M NaCl or CaCl2) or brief wall digestion can enrich activity. Avoid detergents that denature the enzyme or interfere with DNS color development.
• Substrate: Sucrose purity matters; residual glucose/fructose confounds blanks. Run substrate and matrix blanks.
• Linear range: Maintain substrate excess and short incubations to keep initial-rate conditions. Validate linearity with time and protein.
• Interferences: Phenolics and strong reductants reduce DNS non-specifically; consider PVPP in extraction for phenolic-rich tissues and include appropriate controls.
• Standard curve: Construct with glucose (or fructose) standards in the same buffer/conditions as samples.

Reaction Conditions

The following are typical literature conditions for assaying cell wall-bound acid invertase with a DNS micro method. These are general guidelines and not specifications for this item; consult the kit insert for validated parameters.

• Enzyme source: Cell wall-enriched fraction from plant tissue (e.g., salt-extracted apoplastic fraction). Keep samples on ice prior to assay.
• Substrate: Sucrose, 50–200 mM in assay buffer. Ensure minimal glucose/fructose contamination; prepare fresh.
• Buffer: 50–100 mM sodium acetate, pH 4.8–5.2 (acid range where CWI is active). Avoid strong reductants during the detection step.
• Temperature: 30–37°C incubation for 10–30 minutes. Verify linearity with time and enzyme amount.
• Quench/develop: Add DNS color reagent, mix, and heat (e.g., boiling water bath, ~5–10 minutes) to develop the chromophore. Cool to room temperature.
• Measurement: Read absorbance at ~540 nm (exact λmax may vary with formulation). Use matched blanks (substrate blank, sample blank without substrate) to correct background.
• Calibration: Prepare a standard curve using glucose (and/or fructose) across the expected range in the same buffer matrix; fit linear regression within the validated range.
• Calculation: Convert absorbance to μmol reducing sugar using the standard curve. Define 1 unit of CWI activity as 1 μmol of glucose-equivalents released per minute under assay conditions (literature convention; confirm definition in kit insert).
• Common interferences: Phenolics, high salt, detergents, and strong reductants can affect color yield. Use PVPP during extraction and minimize interfering agents.

Safety and Handling

Authoritative safety information is provided in the kit’s SDS and Instructions for Use. The following guidance is general and does not replace the SDS.

Product Data safety fields

• GHS classification: Not specified for this item; refer to CoA/Spec Sheet.
• Signal word / H-statements / Pictograms: Not specified for this item; refer to CoA/Spec Sheet.

General laboratory precautions for DNS-type colorimetric assay kits (literature/good practice)

• Personal protective equipment: Lab coat, nitrile gloves, and splash-resistant safety glasses. Work in a fume hood when heating color-developing reagents.
• Chemical hazards: DNS-type reagents are nitroaromatic oxidants in alkaline solution and can be irritant/corrosive; strong alkali and hot solutions pose burn risks. Avoid inhalation of aerosols and skin/eye contact. Some kits may include metal salts or tartrate; review SDS for specifics.
• Thermal step: Color development typically requires heating (e.g., boiling water bath). Use heat-resistant tubes/racks and avoid sealed vessels when heating. Allow tubes to cool before handling.
• Incompatibilities: Avoid mixing alkaline color reagent with strong acids or oxidizable organics outside the intended protocol. Keep away from reducing agents until assay use.
• Waste: Nitroaromatic-containing waste and strongly alkaline solutions must be collected as hazardous chemical waste following institutional and local regulations.
• First aid (overview): In case of skin/eye contact, rinse with water for at least 15 minutes and seek medical attention as needed. If inhaled, move to fresh air. If ingested, do not induce vomiting; obtain medical attention. Always follow the SDS.

Storage from Product Data

• Store at 2–8°C, protected from light. Shipments on wet ice; maintain cold chain upon receipt.

Solvent Selection

This product is an assay kit; solvent selection in the synthetic sense does not apply. Use the diluents and buffers supplied or recommended in the kit insert.

General guidance (literature)

• Enzymatic step: Cell wall-bound acid invertase exhibits maximal activity in mildly acidic aqueous buffers (e.g., sodium acetate or citrate–phosphate, pH ~4.5–5.5). Organic cosolvents should be minimized to avoid enzyme inhibition.
• Color development: DNS-type reagents are prepared in aqueous alkaline media; heating drives the color-forming reaction between reducing sugars and the reagent. Strictly follow the kit’s buffer recipes to ensure linear response.

If adapting to alternative formats

• For microplate assays, ensure the optical pathlength and well material (polystyrene vs UV-transparent) are compatible with the detection wavelength (typically ~540 nm for DNS). Avoid solvents that may craze or extract plate materials.
• Degassing is generally unnecessary; however, avoid foaming which can cause pathlength variability.

Storage and Reconstitution

From Product Data

• Storage: Store at 2–8°C, protected from light.
• Shipping: Shipped on wet ice.

Kit components

• Exact component list, reconstitution requirements (if any lyophilized materials are supplied), and working solution stability: Not specified for this item; refer to CoA/Spec Sheet.

General handling guidance (not item-specific)

• Upon receipt: Inspect the shipment immediately; keep components refrigerated (2–8°C) and protected from light. Do not freeze unless the kit insert explicitly permits.
• Light sensitivity: Color reagents and standards used in DNS-type methods are typically light sensitive; minimize exposure and store in amber containers when provided.
• Equilibration: Before use, equilibrate reagents to room temperature if directed by the protocol; mix gently to avoid bubbles that affect absorbance readings.
• Aliquoting: If repeated use is expected, aliquot light/air-sensitive reagents to minimize degradation from frequent opening.
• Stability: Observe any expiration date and do not use beyond recommended shelf-life. Discard reagents that show precipitation, discoloration, or microbial growth unless the insert indicates such features are normal.
• Reconstitution water quality: If reconstitution is required, use freshly prepared, high-purity water (e.g., 18 MΩ·cm) to avoid background absorbance or microbial contamination; details to be taken from the kit insert.

Structure and Identity

This product is an assay kit, not a single defined chemical substance. As such, discrete molecular identifiers are not applicable.

• Item type: Cell Wall-Bound Acid Invertase Activity Assay Kit (DNS, Micro Method)
• Composition: Multiple reagents supplied for a colorimetric enzyme assay (kit format). Exact constituents and concentrations are Not specified for this item; refer to CoA/Spec Sheet.
• Molecular formula: Not applicable to a multi-component kit.
• Molecular weight: Not applicable to a multi-component kit.
• SMILES / InChI / InChIKey: Not applicable (mixture/kit); Not specified for this item; refer to CoA/Spec Sheet.
• Structural features (descriptive): The kit measures the hydrolytic activity of cell wall-bound acid invertase (an apoplastic β-fructofuranosidase) on sucrose. Detection relies on the classic “DNS micro method,” wherein reducing sugars generated by the enzyme are quantified colorimetrically via the reduction of a nitroaromatic reagent (literature: 3,5-dinitrosalicylic acid-based assay). No single 2D structure represents the kit.

Synthetic Utility

Not applicable. This is an analytical/biochemical assay kit, not a building block or reagent for chemical synthesis.

General note (context)

• The kit facilitates measurement of enzymatic hydrolysis of sucrose; it is not designed to participate as a reagent or catalyst in preparative transformations.
• If your objective is chemical synthesis of carbohydrates or glycosides, consider dedicated catalysts, enzymes (e.g., glycosyltransferases, invertase in biocatalysis contexts), or protecting-group strategies; this assay kit is solely for quantifying activity in research samples.

Target Specificity

Only information from Product Data may be listed here. No antigen/epitope/clone/isotype or tested specificity parameters are applicable to this assay kit format.

• Target: Cell wall-bound acid invertase activity (enzyme activity in samples), measured via reducing sugar formation using a DNS micro method.
• Detailed target attributes (e.g., isoform specificity, cross-reactivity): Not specified for this item; refer to CoA/Spec Sheet.