Acetylcholine (Ach) Content Assay Kit (Colorimetric Method)

Cat. No.: A1521781

GRAU & PUREZA BioReagent

Visão geral

Acetylcholine (Ach) is the earliest discovered neurotransmitter and an excitatory neurotransmitter for multiple peripheral nerves, including motor nerves, preganglionic fibers of the autonomic nervous system and postganglionic parasympathetic nerve fibers. Ach reacts with substrate solution to form brown compounds via chromogenic reaction; the absorbance (OD value) is measured, and color intensity is positively correlated with Ach concentration.

Kit Composition & Preparation  

A1521781	Components	Appearance	24T/24S	Storage
A1521781A	Extraction Solution	Liquid	30ml	2-8℃.	—
A1521781B	Reagent 1	Solid	1bottle	2-8℃.	1. Tap the bottle to collect all powder at bottom before opening cap; 2. Dissolve powder with 5 mL distilled water before use; 3. Shelf life is consistent with kit expiry date.
A1521781C	Reagent 2	Liquid	5ml	2-8℃.	—
A1521781D	Reagent 3	Liquid	4ml	2-8℃. Store in the dark.	—
A1521781E	Reagent 4A	Solid	3vials	2-8℃. Store in the dark.	1. Before assay, dissolve each vial of Part A with 1.2 mL Part B and mix thoroughly; 2. Shelf life matches kit expiry.
A1521781F	Reagent 4B	Liquid	4ml	2-8℃. Store in the dark.	—
A1521781G	Standard	Solid	1vial	2-8℃. Store in the dark.	1. Required for new standard curve preparation; 2. Prepare stock solution per standard curve protocol in manual; 3. Prepared standard solution shall be used within 7 days.

Working Mix Preparation: Mix Reagent 1 and Reagent 2 at 1:1 volume ratio immediately before experiment to prepare working mix.

Required Lab Equipment

Mortar/homogenizer, ice box/ice maker, bench-top centrifuge, adjustable micropipette, water bath/incubator/dry block heater, 1 mL cuvette, centrifuge tubes, spectrophotometer, distilled water (deionized or ultrapure water acceptable).

Sample Assay Protocol

Pre-test recommendation: Run pilot assay with 1–3 samples of distinct groups to optimize loading volume and avoid unnecessary waste of samples and reagents.

1. Sample Extraction

① Tissue samples: Weigh ~0.1 g tissue, add 1 mL extraction solution, homogenize on ice. Centrifuge at 12000 rpm, 4 ℃ for 10 min, collect supernatant and keep on ice for testing.

Note: For increased loading, extract at tissue weight (g) : extraction solution (mL)=1 : 5~10.

② Cells/Bacteria: Pellet cells/bacteria by centrifugation and discard supernatant; take ~5×10⁶ cells, add 1 mL extraction solution, lyse on ice via sonication (20%/200 W power, 3 s pulse, 10 s interval, repeat 30 cycles). Centrifuge at 12000 rpm, 4 ℃ for 10 min, collect supernatant on ice.

Note: For increased loading, extract at cell count (10⁴) : extraction solution (mL)=500~1000 : 1.

③ Liquid samples: Test directly; centrifuge turbid samples and collect supernatant before detection.

2. Colorimetric Assay

①  Pre-warm spectrophotometer for 30 min and set wavelength to 540 nm.

②  Equilibrate all reagents to room temperature (25 ℃), dispense into EP tubes as below:

Reagent (μL)	Assay tube	Blank tube
Sample	100	—
Water	200	300
Working Mix	300	300

Mix and incubate at RT for 15 min

Reagent (μL)	Assay tube	Blank tube
Reagent 3	150	150
Reagent 4	100	100

Vortex EP tube thoroughly for 2 min, transfer entire clear supernatant into 1 mL glass cuvette (1 cm path length), read absorbance at 540 nm. ΔA=A_assay-A_blank ;

Notes:

1. If ΔA is too low, raise sample volume V1 from 100 μL to 200 μL with corresponding reduction in water volume; update V1 in calculation formula.
2. Keep ΔA within linear range of standard curve. If ΔA＞1.2, dilute sample and record dilution factor D for calculation, or reduce V1 (e.g. to 50 μL, add more water); substitute updated V1 and D into formula.

Calculation Formulas

1. Standard curve: y = 6.2507x - 0.0252; x=standard mass (mg), y=ΔA

2. By tissue weight: Ach content (mg/g tissue)=[(ΔA+0.0252)÷6.2507]÷(W×V1÷V)×D=1.6×(ΔA+0.0252)÷W×D

3. By total protein: Ach content (mg/mg protein)=[(ΔA+0.0252)÷6.2507]÷(Cpr×V1÷V)×D=1.6×(ΔA+0.0252)÷Cpr×D

4. By cell count: Ach content (μg/10⁴ cells)=[(ΔA+0.0252)÷6.2507×10³]÷(500×V1÷V)×D=1600×(ΔA+0.0252)÷500×D

5. By liquid volume: Ach content (mg/mL)=[(ΔA+0.0252)÷6.2507]÷V1×D=1.6×(ΔA+0.0252)×D

Parameter Definition:

W=sample weight (g);

V=total extraction volume (1 mL);

V1=supernatant loading volume (0.1 mL);

500=cell count (×10⁴);

D=dilution factor (D=1 without dilution);

Cpr=sample protein concentration (mg/mL); BCA protein quantification kit from our company is recommended.

Appendix: Standard Curve Preparation

1. Weigh 2 mg standard powder into EP tube, dissolve in 1 mL water to prepare 2 mg/mL stock solution (prepare fresh, use within 48 h). Serially dilute stock to 6 concentrations: 0, 0.4, 0.8, 1.2, 1.6, 2 mg/mL (adjust concentration range per actual sample).

1. Weigh 2 mg standard powder into EP tube, dissolve with 1 mL distilled water to prepare 2 mg/mL standard stock solution (prepare freshly, use within 2 days). Serially dilute stock solution with distilled water to six gradient concentrations: 0, 0.4, 0.8, 1.2, 1.6, 2 mg/mL. Adjust concentrations according to actual sample condition if needed.

2. Standard dilution table:

Std Conc.(mg/mL)	0	0.4	0.8	1.2	1.6	2
Std stock (μL)	0	40	80	120	160	200
Distilled water (μL)	200	160	120	80	40	0

Mix all standard tubes ready for use.

3. Dispense reagents following table below, calculate ΔA by subtracting absorbance of zero-concentration blank, then plot zero-based standard curve.

Reagent (μL)	Assay tube	Zero-concentration blank (run once only)
Standard	100	—
Distilled water	200	300
Working Mix	300	300

Incubate at RT for 15 min

Reagent (μL)	Assay tube	Zero-concentration blank (run once only)
Reagent 3	150	150
Reagent 4	100	100

Vortex EP tube for 2 min, transfer all clear liquid to 1 cm-path 1 mL cuvette, read OD at 540 nm: ΔA=A_assay-A_{zero blank}

Especificações

Sinónimos

Acetylcholine (ACH) assay kit

Especificações e pureza

BioReagent

Grau

BioReagent

Aplicação

Cell Metabolism

Tipo de amostra

Bacteria, Cell, Tissue

Método de deteção

Colorimetric Method

Detection instrument

Visible Spectrophotometer

Detection wavelength

540 nm

Sensibilidade

Light-sensitive

Armazenamento e envio

Condições de armazenamento de armazenamento

Store at 2-8°C,Protected from light

Enviado em

Wet ice

Estabilidade e armazenamento

Store at 2-8℃ long term (3 months). Store in the dark.

Contents & Storage

A1521781	Components	Appearance	24T/24S	Storage
A1521781A	Extraction Solution	Liquid	30ml	2-8℃.
A1521781B	Reagent 1	Solid	1bottle	2-8℃.
A1521781C	Reagent 2	Liquid	5ml	2-8℃.
A1521781D	Reagent 3	Liquid	4ml	2-8℃. Store in the dark.
A1521781E	Reagent 4A	Solid	3vials	2-8℃. Store in the dark.
A1521781F	Reagent 4B	Liquid	4ml	2-8℃. Store in the dark.
A1521781G	Standard	Solid	1vial	2-8℃. Store in the dark.