Acetylcholinesterase (AchE) activity detection kit (DNTB, microcalorimetry)

Cat. No.: A1506759

GRADE & PURITY BioReagent

Overview

  Acetylcholinesterase (AChE) is a serine hydrolase primarily found in the nervous system. It exists widely in various animal tissues and serum in multiple isoforms. AChE catalyzes the hydrolysis of the neurotransmitter acetylcholine, thereby terminating cholinergic nerve signal transmission. It plays a crucial role in the regulation of neural conduction, and also functions in inducing axon growth, promoting synapse formation, and facilitating hematopoietic cell formation.

Detection Principle

  AChE catalyzes the hydrolysis of acetylcholine (Ach) to produce choline. Choline reacts with 5,5'-Dithio-bis-(2-nitrobenzoic acid) (DTNB) to generate 5-thio-2-nitrobenzoic acid (TNB). TNB has an absorption peak at 412 nm. The AChE activity is calculated by determining the rate of increase in absorbance at 412 nm.

Applicable Samples: Fresh serum (plasma), animal tissues, nerve cells.

A1506759	Component	96T	Storage
A1506759A	Extraction Buffer	120 mL	2-8℃
A1506759B	Assay Buffer	20 mL	2-8℃
A1506759C	Chromogen	1.3 mL	2-8℃. Store in the dark.
A1506759D	Substrate	1.3 mL	2-8℃. Store in the dark.

Note: Before formal testing, it is recommended to perform a preliminary assay using 2-3 samples expected to have significant differences.

Materials Required but Not Provided

1. Microplate reader or visible spectrophotometer (capable of measuring absorbance at 412 nm)

2. Incubator, ice maker, refrigerated centrifuge

3. 96-well plate or micro glass cuvettes, adjustable pipettes, and tips

4. Deionized water

5. Homogenizer (if processing tissue samples)

Experimental Procedure

1. Reagent Preparation

Reagent Name	Preparation	Notes
Extraction Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Assay Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Chromogen	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C protected from light. Irritant. Wear appropriate personal protective equipment.
Substrate	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C protected from light.
Working Solution	Mix Assay Buffer, Chromogen, and Substrate uniformly in a 16:1:1 ratio.	Prepare immediately before use.

2. Sample Preparation

2.1 Animal Tissue

Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize in an ice bath. Centrifuge at 8,000 × g, 4°C for 10 minutes. Collect the supernatant and place it on ice for assay.

2.2 Nerve Cells

Collect 5 million cells into a centrifuge tube. Wash the cells with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt the cells using an ultrasonic cell disruptor in an ice bath for 5 minutes (20% power or 200 W, ultrasound for 3 s, interval for 7 s, repeat 30 times). Then centrifuge at 8,000 × g, 4°C for 10 minutes. Collect the supernatant and place it on ice for assay.

2.3 Fresh Serum (Plasma) and Other Liquid Samples

Assay directly.

Notes:

① Fresh samples are recommended. If not assayed immediately, samples can be stored at -80°C for up to 1 month.

② For animal tissues with high fat content, remove the upper fat layer after centrifugation before collecting the supernatant.

③ If measuring protein concentration, the Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.

3. Assay Procedure

3.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 412 nm. Zero the visible spectrophotometer with deionized water.

3.2 Preheat the incubator to 37°C. Pre-warm the Working Solution in the incubator for 30 minutes.

3.3 Add 20 μL of sample and 180 μL of pre-warmed Working Solution into a 96-well plate or micro glass cuvette. Mix rapidly and incubate at 37°C for 3 minutes. Monitor the absorbance change at 412 nm over 3 minutes. Record the absorbance at 20 seconds as A₁ and the absorbance at 200 seconds as A₂. Calculate ΔA = A₂ - A₁.

Note: Preliminary tests with 2-3 samples expected to have significant differences are recommended. If ΔA is less than 0.002, consider increasing the sample volume appropriately. If ΔA is greater than 0.8, the sample can be further diluted with Extraction Buffer (multiply the result by the dilution factor) or the sample amount used for extraction can be reduced.

4. Calculation of Results

Note: We provide both a detailed derivation formula and a simplified formula. They are exactly equivalent. The simplified formula (in bold) is recommended as the final calculation formula.

4.1 Calculation Formulas for 96-Well Plate Assay

(1) AChE Activity in Animal Tissue

A. Calculation based on protein concentration

Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 nmol of TNB per minute per mg of protein.

AChE Activity (U/mg prot)

= (ΔA ÷ ε ÷ d × V_{reaction total} × 10⁹) ÷ (Cpr × V_sample) ÷ T 

= 490 × ΔA ÷ Cpr × n B. Calculation based on sample weight 

Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 nmol of TNB per minute per gram of tissue. AChE Activity (U/g fresh weight) 

= (ΔA ÷ ε ÷ d × V_{reaction total} × 10⁹) ÷ (W × V_sample ÷ V_{sample total}) ÷ T 

= 490 × ΔA ÷ W × n 

(2) AChE Activity in Nerve Cells 

Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 nmol of TNB per minute per 10⁴ cells. AChE Activity (U/10⁴ cells) 

= (ΔA ÷ ε ÷ d × V_{reaction total} × 10⁹) ÷ (Cell Number × V_sample ÷ V_{sample total}) ÷ T 

= 490 × ΔA ÷ 500 × n = 0.98 × ΔA × n 

(3) AChE Activity in Serum (Plasma) and Other Liquid Samples 

Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 nmol of TNB per minute per mL of sample. AChE Activity (U/mL) 

= (ΔA ÷ ε ÷ d × V_{reaction total} × 10⁹) ÷ V_sample ÷ T 

= 490 × ΔA × n 

Parameter Description: 

ε: Molar extinction coefficient of TNB, 13.6 × 10³ L/mol/cm 

d: Light path for the 96-well plate, 0.5 cm 

V_{reaction total}: Total reaction volume, L; 

200 μL = 2 × 10⁻⁴ L 

10⁹: Conversion factor from moles to nmoles; 

1 mol = 1 × 10⁹ nmol 

Cpr: Protein concentration, mg/mL 

V_sample: Volume of supernatant added, 0.02 mL 

T: Reaction time, 3 min 

n: Sample dilution factor 

W: Sample weight, g 

V_{sample total}: Volume of extraction buffer, 1 mL 

500: Total cell number, 5 million (500 × 10⁴)

4.2 Calculation Formulas for Micro Glass Cuvette Assay

Adjust the light path d in the formulas above from 0.5 cm to 1 cm and calculate accordingly.

Precautions

This product is for research use only. Not for use in diagnostic procedures. For your safety and health, please wear lab coats and disposable gloves during operation.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.