Description
Acetylcholine Assay Kit (BA0069) (BA0069)
The Acetylcholine Assay Kit (SKU: BA0069) provides a simple, direct and high-throughput method for measuring acetylcholine in biological samples. Acetylcholine is a neurotransmitter produced in acetylcholinergic neurons that plays important roles in skeletal muscle movement, regulation of smooth and cardiac muscles, and in learning, memory and mood. In this assay, acetylcholine is hydrolysed by acetylcholinesterase to choline, which is oxidised by choline oxidase to betaine and hydrogen peroxide. The resulting hydrogen peroxide reacts with a specific dye to form a pink coloured product. The colour intensity at 570 nm, or fluorescence intensity at excitation/emission 530/585 nm, is directly proportional to the acetylcholine concentration in the sample.
| Product Name: | Acetylcholine Assay Kit (BA0069) |
| SKU: | BA0069 |
| Detection Method: | Colorimetric or Fluorometric |
| Detection Range: | Colorimetric 10 to 200 uM; fluorometric 0.4 to 10 uM acetylcholine |
| Sample Type: | Serum, plasma, urine, saliva, milk, tissue and cell culture |
| Species Reactivity: | All |
| Assay Time: | 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
Quantitative colorimetric or fluorometric determination of acetylcholine. Acetylcholine is hydrolysed to choline, which is oxidised to produce hydrogen peroxide that reacts with a dye read at 570 nm or fluorometrically at excitation/emission 530/585 nm.
- Uses 20 uL sample
- Colorimetric linear detection range 10 to 200 uM acetylcholine
- Fluorometric linear detection range 0.4 to 10 uM acetylcholine
- Simple, direct and high-throughput format
- Assays of acetylcholine in serum, plasma, urine, saliva, milk, tissue and cell culture
- Drug discovery and pharmacology: effects of drugs on acetylcholine metabolism
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Sample treatment. Serum and plasma can be assayed directly. Prepare tissue and cell lysates by homogenising in cold 1 x PBS and centrifuging for 5 minutes at 14,000 rpm; use clear supernatant. Clear milk samples by mixing 600 uL milk with 100 uL 6 N HCl, centrifuging, then neutralising 300 uL supernatant with 50 uL 6 N NaOH (dilution factor n = 1.36). |
| 2 | Reconstitution. Equilibrate components to room temperature, briefly centrifuge tubes and keep thawed tubes on ice. Reconstitute Enzyme Mix with 120 uL Assay Buffer; it is stable for 1 month at -20C. If a yellow precipitate forms, centrifuge and use the clear supernatant. |
| 3 | Standards. Mix 24 uL 2 mM Standard with 216 uL distilled water (final 200 uM) and prepare the dilution series shown in the table. Transfer 20 uL diluted standards into separate wells of a clear flat-bottom 96-well plate. Transfer 20 uL of each sample into separate wells; for samples known to contain choline, prepare an extra Sample Blank well. |
| 4 | Colour reaction. Prepare working reagent by mixing, for each well, 85 uL Assay Buffer, 1 uL ACHE Enzyme, 1 uL Enzyme Mix and 1 uL Dye Reagent (omit ACHE Enzyme for Sample Blanks). Add 80 uL working reagent to each well, tap to mix and incubate 30 minutes at room temperature. |
| 5 | Read optical density at 570 nm (550-585 nm). For the fluorometric assay use 0, 3, 6 and 10 uM standards and a black plate, reading fluorescence at excitation 530 nm and emission 585 nm. |
Subtract the blank value (standard #4) from the standard values and plot the change in OD or fluorescence against standard concentrations. Determine the slope and calculate [Acetylcholine] = (Rsample - Rblank) / slope x n, where n is the sample dilution factor. Conversion: 1 mM acetylcholine equals 14.6 mg/dL.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| ACHE Enzyme | 120 uL | -20C |
| Enzyme Mix (dried) | 1 vial | -20C |
| Dye Reagent | 120 uL | -20C |
| Standard (2 mM acetylcholine) | 400 uL | -20C |
