Glycogen Phosphorylase b (GPb) Activity Assay Kit (UV Micro Method)

Cat. No.: G1521925

GRADE & PURITY BioReagent

Visão geral

Glycogen phosphorylase (GP, EC 2.4.1.1) is a key enzyme in glycogen catabolism. It cleaves α-1,4-glycosidic bonds one by one from the non-reducing ends of the glycogen molecule, removing glucosyl residues and releasing glucose-1-phosphate, until it reaches four glucose residues before the α-1,6-glycosidic branch points. GP exists in two forms: the active glycogen phosphorylase a (GPa) and the inactive glycogen phosphorylase b (GPb). GPb can be activated in the presence of a certain concentration of adenylate (5'-AMP). Determination Principle: GP catalyzes the reaction between glycogen and inorganic phosphate, producing a shortened glycogen molecule and glucose-1-phosphate. Phosphoglucomutase and glucose-6-phosphate dehydrogenase subsequently catalyze the reduction of NADP⁺ to NADPH. GP activity is determined by measuring the rate of increase in NADPH at 340 nm. GP (GPa and GPb) activity is measured in the presence of a certain concentration of adenylate (5'-AMP), while GPa activity is measured in the absence of adenylate (5'-AMP). GPb activity is obtained by subtracting GPa activity from total GP activity.

Specifications

Especificações e pureza

BioReagent

Grau

BioReagent

Aplicação

Enzyme activity assay

Detection instrument

Microplate reader,UV Spectrophotometer

Detection wavelength

340nm

Armazenamento e envio

Condições de armazenamento de armazenamento

Store at 2-8°C

Enviado em

Wet ice

Estabilidade e armazenamento

Store at 2-8℃ for 6 months.

Application Protocols

• Item-specific protocols

  • A validated step‑by‑step protocol, reagent preparation instructions, and plate layout are not specified for this item; refer to the kit’s instruction booklet.

• General microplate UV GPb assay workflow (literature; not item‑specific)

  1. Equilibrate all reagents to assay temperature; prepare fresh working solutions of substrate(s), buffer, cofactors, and coupling enzymes.
  2. Prepare sample dilutions to place initial rates in a linear, measurable window (e.g., ΔA340/min between 0.01–0.3, depending on reader sensitivity).
  3. Dispense master mix into wells; add samples or standards (keep DMSO ≤1–2% if used for inhibitor solubilization, and include vehicle controls).
  4. Initiate reaction (e.g., by adding glycogen or enzyme). Mix gently, avoid bubbles.
  5. Read A340 at 10–20 s intervals for 5–10 min. Verify linearity over the acquisition window.
  6. Calculate slopes (ΔA/min), subtract blanks, and convert to activity using pathlength‑corrected extinction coefficients. Normalize to protein content (e.g., BCA assay) if comparing lysates.
  7. For inhibitor testing: perform ≥8‑point dose–response with duplicates/triplicates; fit to a four‑parameter logistic model to estimate IC50.

• Tips

  • Use UV‑transparent plates; calibrate plate reader pathlength. Keep phosphate concentration consistent across wells. Include internal controls across plates to track inter‑run variability.

Biological Roles

• General biology of GPb (literature; not item‑specific)

  • Function: Glycogen phosphorylase b (EC 2.4.1.1) catalyzes the phosphorolytic cleavage of glycogen to produce glucose‑1‑phosphate (G1P), a key step in glycogenolysis.
  • Regulation: Exists in two interconvertible forms—b (less active) and a (phosphorylated, more active). Allosteric regulators include AMP (activator), ATP and glucose‑6‑phosphate (inhibitors). PLP is an essential cofactor.
  • Localization and isoforms: Isoforms are expressed in muscle (PYGM), liver (PYGL), and brain (PYGB). Subcellular localization is predominantly cytosolic with association to glycogen granules.
  • Pathway context: Couples to glycolysis and the pentose phosphate pathway via conversion of G1P to G6P (by phosphoglucomutase), supporting ATP generation and NADPH production depending on cellular demands.
  • Research relevance: GP regulation is a central model for studying allostery, phosphorylation cascades, and energy metabolism. GPb activity measurements inform studies on exercise physiology, metabolic regulation, and enzymology of carbohydrate metabolism.

• Notes for assays (general)

  • Ionic strength, pH (often near 6.8–7.4), and inorganic phosphate concentration critically modulate GPb kinetics. AMP can be used to probe allosteric activation in vitro.
  • Sample‑specific confounders: Endogenous dehydrogenases, phosphatases, or NAD(P)H background in lysates may affect 340 nm signals; appropriate blanks and desalting steps are recommended.

Buffer Applications

This product is an enzyme activity assay kit rather than a standalone buffer. Nonetheless, buffer selection is central to GPb activity measurements.

• Item-specific details

  • Specific buffer composition and pH are not specified for this item; refer to the kit insert.

• General guidance (literature; not item‑specific)

  • pH range: GPb shows robust activity near neutral pH; assays are commonly run around pH 6.8–7.4 using buffers such as HEPES, MOPS, or phosphate. Note that high phosphate both buffers and serves as a substrate; maintain controlled concentrations to avoid confounding kinetics.
  • Common components: Glycogen (substrate), inorganic phosphate, PLP (cofactor), Mg2+ (for coupling enzymes), and AMP (allosteric activator) may be present in assay mixtures depending on the specific format.
  • Ionic strength: 50–150 mM total salt is typical for enzyme stability; optimize to balance activity and coupling efficiency.
  • UV compatibility: Choose buffers with minimal absorbance at 340 nm (e.g., HEPES over Tris at high concentrations). Avoid reducing agents or detergents that interfere with dehydrogenase readouts unless validated.
  • Controls: Include no‑enzyme and no‑substrate controls, and a vehicle control if screening inhibitors dissolved in DMSO.

• Preparation tips

  • Filter (0.22 µm) and degas buffers to improve baseline stability; pre‑equilibrate to assay temperature before use.

Green Alternatives

• Context

  • Historic assays for glycogen phosphorylase included radiometric methods or endpoint colorimetric assays with larger reagent volumes. A UV microplate method minimizes hazardous waste and sample consumption.

• Greener aspects of a UV micro method (general; not item‑specific)

  • Microvolume format: Lower reagent consumption and reduced chemical exposure.
  • Aqueous buffers: Avoidance of organic solvents common in some analytical workflows.
  • Non‑radioactive detection: Eliminates radiological hazards and specialized waste streams.

• Comparison (general)

  • Radiometric GP assays vs UV micro method
    • Safety: UV method avoids radioisotopes.
    • Waste: Sharply reduced hazardous waste volume.
    • Throughput: Microplates enable higher throughput and replicate density.
    • Sensitivity: Radiometric methods can be very sensitive; UV methods rely on NADPH Δε340 ≈ 6.22 mM−1 cm−1 (literature) and may require optimization for low‑activity samples.

• Best practices for greener labs

  • Right‑size reaction volumes (96‑ or 384‑well plates), optimize to minimize excess reagents, and segregate aqueous waste. Use reusable reservoirs and low‑adsorption tips to reduce plastic waste without compromising assay integrity.

Pharmaceutical Uses

This is a research‑use enzyme assay kit and is not intended for clinical diagnostics, therapeutic, or manufacturing use.

• Item-specific status

  • Research Use Only (RUO): As stated in Product Data. No pharmacopeial monograph or excipient role is provided for this item.

• General research utility (not item‑specific)

  • Preclinical discovery: RUO GPb assays are commonly used to profile enzyme kinetics, evaluate small‑molecule modulators, and characterize mechanism of action in biochemical assays.
  • ADME/assay support: When screening potential drug candidates that target carbohydrate metabolism, biochemical potency against GPb can complement cell‑based assays. Any translation to regulated environments requires separate, validated kits and methods compliant with GLP/GMP—outside the scope of this RUO product.

Physical Properties

This is a reagent kit comprising multiple components (buffers, substrates, cofactors, and/or coupling enzymes). A single set of physical constants does not apply.

• Item-specific physical specifications

  • Boiling point, melting point, density, refractive index, pKa, logP, UV cutoff: Not specified for this item; refer to CoA/Spec Sheet for component‑level details, if provided.
  • Appearance: Not specified for this item; refer to CoA/Spec Sheet.

• General considerations (literature; not item‑specific)

  • Assay solutions are typically aqueous buffers formulated near neutral pH to maintain GPb activity and stability.
  • UV micro method relies on monitoring NAD(P)H absorbance at 340 nm; thus, components are chosen to be compatible with UV detection and low background at 340 nm.
  • Working solutions are commonly prepared fresh and kept chilled during setup to preserve enzymatic activity; minimize exposure to strong light if NAD(P)+/NADP+ or NADH/NADPH is involved.

Quality and Grades

• Item-specific grade

  • BioReagent: Indicates suitability for general biochemical/biological research workflows. Typical expectations include component quality compatible with enzyme assays and spectrophotometric readouts. Exact assay performance metrics (e.g., LOD/LOQ, dynamic range) are not specified for this item; refer to CoA/Spec Sheet or kit insert.

• What BioReagent means in practice (general guidance)

  • Low background at 340 nm for UV enzyme kinetics and adequate purity of substrates/cofactors to minimize side reactions.
  • Buffers and salts prepared to research grade, with typical control of trace contaminants (metals, peroxides) that could inactivate enzymes or bias readouts. Specific contaminant limits are not specified for this item.

• Stabilizers and preservatives

  • Not specified for this item; refer to CoA/Spec Sheet. If present, stabilizers extend shelf life and activity of enzymes; preservatives deter microbial growth in aqueous components.

• Documentation and QC

  • For batch‑specific acceptance criteria (e.g., reference rate curve, control activity recovery), consult the kit’s Certificate of Analysis and instruction booklet. Always include provided controls or recommended standard curves to verify lot performance.

Reaction and Applications

• Item-specific intent

  • Designed to quantify Glycogen Phosphorylase b (GPb) activity using a UV microplate readout. Detailed component list and reaction scheme are not specified for this item; refer to the kit insert.

• Assay principle (literature; typical UV micro method, not item‑specific)

  • GPb catalyzes phosphorolysis of glycogen to glucose‑1‑phosphate (G1P). Activity is often coupled: G1P → G6P via phosphoglucomutase (PGM), then G6P + NADP+ → 6‑phosphoglucono‑δ‑lactone + NADPH via glucose‑6‑phosphate dehydrogenase (G6PD). NADPH formation is monitored at 340 nm in microplates.
  • Alternative coupling uses phosphogluconate dehydrogenase or hexokinase/glucose‑6‑phosphate dehydrogenase systems. The UV micro method provides continuous kinetics suitable for V0 rate determination.

• Typical research applications (literature)

  • Enzyme kinetics: Determine Km for glycogen or phosphate, Vmax, kcat, and allosteric modulation by AMP/ATP/G6P.
  • Inhibitor screening: IC50/Ki estimation for small‑molecule GPb inhibitors; vehicle control required for cosolvents.
  • Sample testing: Measure GP activity in tissue/cell lysates (e.g., muscle, liver) after suitable sample prep and desalting to remove interfering phosphatases/oxidants.
  • Comparative biology: Assess GPb vs GPa states under different phosphorylation conditions using phosphatase/kinase treatments upstream.

• Practical tips (general)

  • Maintain constant temperature (e.g., 25–37°C) and pathlength correction for microplates. Pre‑equilibrate buffers, confirm linearity of rate with enzyme concentration, and include a no‑enzyme blank to account for background at 340 nm.

Reaction Conditions

• Item-specific instructions

  • Exact reagent compositions, volumes, incubation times, and controls are not specified for this item; refer to the kit’s instruction booklet for validated conditions.

• General conditions for UV microplate GPb assays (literature; not item‑specific)

  • Temperature: 25–37°C, held constant (±0.5°C). Many labs use 30°C for enzyme kinetics or 37°C for physiological relevance.
  • Detection: Measure ΔA340/min for NADPH formation in clear, UV‑transparent plates; pathlength correction may be necessary. Read interval: 10–20 s for 5–10 min to capture initial linear rates.
  • Typical components: Glycogen (0.1–2 mg/mL), inorganic phosphate (1–10 mM), PLP (10–100 µM), AMP (0–1 mM), coupling enzymes (PGM, G6PD), Mg2+ (1–5 mM). Concentrations are literature examples and must be optimized for your system.
  • Buffer: HEPES/MOPS/Tris or phosphate, pH ~6.8–7.4. Avoid high background at 340 nm. Maintain ionic strength (50–150 mM NaCl or KCl) as needed.
  • Enzyme input: Choose GPb concentration that yields a linear rate with respect to time and enzyme amount; verify linearity and avoid substrate depletion.
  • Controls and calibration: Include blank (no enzyme), negative control (no substrate), and positive control (known GPb source or standard). Prepare a calibration using known NADPH to validate A340 → concentration conversion (Δε340 ≈ 6.22 mM−1 cm−1; literature) if pathlength is known.
  • Data analysis: Convert slopes (ΔA/min) to activity (µmol/min) using Beer–Lambert law corrected for well pathlength; normalize to sample mass, protein, or volume.

Safety and Handling

• Item-specific hazard data

  • Signal word, H‑statements, GHS classification, pictograms: Not specified for this item; consult the SDS for each component in the kit.

• General guidance (not item-specific; defer to SDS as authoritative)

  • Personal protective equipment: Use lab coat, safety glasses, and appropriate chemically resistant gloves. Handle in a well‑ventilated area or fume hood when preparing reagents or handling powders.
  • Avoid inhalation of dusts/aerosols from lyophilized components; avoid skin/eye contact with buffers and cofactors. Wash thoroughly after handling.
  • Incompatibilities: Keep enzymatic reagents away from strong acids/bases, oxidizers, and metal contaminants that can inactivate proteins or degrade cofactors. Do not mix with sodium hypochlorite when amines or phosphate buffers are present.
  • First aid (overview): If on skin/eyes, rinse with water for several minutes; remove contaminated clothing. If inhaled, move to fresh air. If ingested, rinse mouth. Seek medical attention as needed. Provide SDS to responders.
  • Special risks: Some enzyme assay kits may include reducing agents (e.g., DTT) or preservatives (e.g., sodium azide) in small quantities—both require careful handling and waste segregation. Do not allow azide‑containing solutions to contact heavy metals to prevent metal azide formation. Note: Composition for this item is not specified; consult the SDS.
  • Waste: Collect aqueous assay waste according to institutional rules for chemical/biological waste. Do not dispose to sink without authorization.

Solvent Selection

This product is supplied as an aqueous enzyme activity assay kit; solvent selection is typically not a variable in routine use.

• Item-specific guidance

  • Use components as supplied or reconstitute with water/buffer specified in the kit insert. Organic solvents can denature enzymes and are generally avoided unless explicitly permitted.

• General considerations (literature; not item‑specific)

  • Polarity: Enzymatic GPb assays are run in aqueous buffers near neutral pH. Dielectric properties of water (ε ~78 at 25°C) support ionic substrates/cofactors.
  • Additives: Small percentages of benign cosolvents (e.g., ≤1–2% DMSO) may be tolerated when screening hydrophobic inhibitors; however, tolerance varies and must be validated with controls. For this item, allowable cosolvent levels are not specified; consult the kit documentation.
  • Alternatives: If organic‑rich conditions are required (e.g., to solubilize test compounds), consider pre‑dissolving in DMSO and performing serial dilutions to keep final DMSO low, with a matching vehicle control in all wells.

Storage and Reconstitution

• Item-specific storage

  • Store at 2–8°C (from Product Data). Shipped on wet ice. Do not freeze unless explicitly permitted by the kit insert. Protect light‑sensitive components from light.

• Stability and handling

  • Working solutions of enzymes/cofactors are generally less stable than concentrates; prepare immediately before use and keep on ice until assay start. Avoid repeated freeze–thaw of any protein components by aliquoting if freezing is allowed.

• Reconstitution

  • Specific reconstitution solvents/volumes for individual components are not specified for this item; refer to the kit insert or CoA. If lyophilized reagents are included, reconstitute with nuclease‑/protease‑free water or the provided buffer, mixing gently to avoid foaming.

• Documentation

  • Record lot numbers, preparation dates, and storage conditions for each component. Inspect for precipitates, turbidity, or color changes before use and discard any compromised reagents following institutional chemical/biological waste policies.

Structure and Identity

This product is a multi‑component enzymatic activity assay kit designed to quantify Glycogen Phosphorylase b (GPb) activity via a UV microplate format. It is not a single chemical entity and therefore has no single molecular structure.

• Item-specific facts (from Product Data)

  • SKU: G1521925
  • Product name: Glycogen Phosphorylase b (GPb) Activity Assay Kit (UV Micro Method)
  • Grade: BioReagent; For research use only
  • Storage: 2–8°C; shipped on wet ice
  • CAS, CID, InChIKey, SMILES: Not applicable to a kit; not specified for this item; refer to CoA/Spec Sheet.
  • Molecular formula / molecular weight: Not applicable to a kit; not specified for this item; refer to CoA/Spec Sheet.

• General background (literature; not item‑specific)

  • Target enzyme: Glycogen phosphorylase b (GPb) is the dephosphorylated form of glycogen phosphorylase (EC 2.4.1.1), typically a homodimeric enzyme that catalyzes phosphorolysis of glycogen to glucose‑1‑phosphate (G1P).
  • Cofactor: Requires pyridoxal 5′‑phosphate (PLP) as a tightly bound cofactor (literature).
  • Regulatory features: GPb is allosterically activated by AMP and inhibited by ATP and glucose‑6‑phosphate; conversion to the “a” form occurs via phosphorylation by phosphorylase kinase (literature).

• Conceptual 2D description (literature)

  • The assay monitors GPb’s conversion of glycogen + Pi → G1P, typically coupled to downstream enzymes that convert G1P to NADPH, enabling UV detection at 340 nm in microplates.

Synthetic Utility

Not typically applicable. This product is an enzymatic activity assay kit and is not used as a synthetic reagent or building block.

• Guidance
  • For information relevant to experimental use, see Reaction & Applications and Reaction Conditions sections, which address kinetic measurements and assay setup rather than chemical synthesis.

Target Specificity

This product is an activity assay kit rather than an antibody or affinity reagent. Antigen/epitope and species reactivity parameters do not apply.

• Item-specific note

  • The functional target is the enzyme Glycogen Phosphorylase b (GPb). Species/isoform specificity and validated sample types are not specified for this item; consult the kit insert for any demonstrated use cases or controls.

• General background (literature)

  • GPb activity can be measured from purified enzyme or from tissue/cell lysates containing PYGM, PYGL, or PYGB isoforms. Assay specificity is achieved biochemically via substrate/coupling design and controls (e.g., omission of glycogen or inorganic phosphate) rather than immuno‑recognition.