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| Product Name | Acetylcholinesterase (AChE) Activity Colorimetric Assay Kit |
| Catalog No. | ETR-HMM-0058 |
| Description | A colorimetric assay for measuring acetylcholinesterase and cholinesterase enzyme activities in biological samples based on the Ellman method. AChE cleaves acetylthiocholine iodide, and the released thiocholine reacts with DTNB (5,5'-dithio-bis(2-nitrobenzoic acid)) to produce the yellow-colored thionitrobenzoate anion measured at 412 nm. |
| Intended Use | Quantitative measurement of AChE activity in brain and nerve tissue homogenates, erythrocytes, whole blood, and neuronal cell culture lysates for neurotoxicology studies, organophosphate pesticide toxicity research, and Alzheimer's disease drug development. |
| Principle / Technology | AChE catalyzes the hydrolysis of acetylcholine (and the synthetic substrate acetylthiocholine) at cholinergic synapses; the released thiocholine reacts with the chromogenic reagent DTNB to produce 2-nitro-5-thiobenzoate (TNB) with a bright yellow color and molar absorbance coefficient of 13,600 M-1cm-1 at 412 nm; the rate of color development is directly proportional to AChE activity. |
| Detection Method | Colorimetric kinetic absorbance measurement at 412 nm using a microplate spectrophotometer; kinetic mode recommended over endpoint for accurate activity determination |
| Sample Type | Brain homogenates (cerebrum, cerebellum, brainstem), erythrocyte lysates, whole blood (diluted), spinal cord tissue, neuronal cell lysates, purified recombinant AChE from electric eel or human |
| Performance Range / Specifications | AChE activity range: 0.01–100 nmol/min/μg protein; AChE standard for calibration included; one unit is defined as the amount cleaving 1 μmol acetylcholine per minute at 25°C; kinetic linear range at least 20 minutes |
| Sensitivity / LOD | Detection of AChE activity in as little as 5 μg total brain protein lysate per reaction in 96-well format |
| Specificity | Acetylthiocholine substrate is cleaved preferentially by AChE (true cholinesterase) over BuChE (pseudocholinesterase or butyrylcholinesterase); selective inhibitors (eserine/physostigmine for AChE, iso-OMPA for BuChE) can be added to distinguish the two enzyme classes; organophosphate compounds inhibit both AChE and BuChE dose-dependently |
| Reaction Conditions / Protocol | Prepare brain or tissue lysates (0.1% Triton X-100 in PBS, homogenize on ice, centrifuge 600 × g); dilute lysates in Assay Buffer; add 160 μL Assay Buffer to well; add 20 μL DTNB Reagent; add 20 μL diluted sample or controls; initiate reaction by adding 10 μL Acetylthiocholine Substrate (75 mM); mix briefly; immediately begin kinetic reading at 412 nm every 1 minute for 15–30 minutes at 25°C; calculate initial reaction rate (V0) from the linear portion of the time course; convert to enzyme activity units using molar extinction coefficient of TNB (13,600 M-1cm-1) |
| Components / Formulation | Acetylthiocholine Iodide Substrate, DTNB Reagent (Ellman's reagent, 10 mM in buffer), Assay Buffer (sodium phosphate, pH 7.0, with BSA stabilizer), AChE Positive Control (electric eel AChE, lyophilized), Eserine inhibitor control (0.5 mM in buffer), detailed kinetic analysis protocol |
| Storage Conditions | Lyophilized AChE at -20°C protected from light; DTNB sensitive to moisture and oxidation, store desiccated at -20°C, prepare fresh working solution; substrate at -20°C; Assay Buffer at 2–8°C |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 200 assays, 500 assays (96-well kinetic format) |
| Product Form | Lyophilized enzyme substrates and controls with liquid assay buffer |
| Quality Control | Each lot tested with electric eel AChE reference: specific activity (nmol/min) within defined range; DTNB purity ≥98%; inhibition by 0.1 mM eserine >90% |
| Key Features | Ellman's method is the gold standard for AChE activity measurement with decades of published data for reference comparison; kinetic measurement mode avoids endpoint timing variability; sensitive enough for ex vivo tissue and blood samples; eserine inhibitor control validates AChE-specific signal |
For research use only, not for clinical use.
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