Description
Acetylcholinesterase Inhibition Assay Kit (Colorimetric) (BA0160) (BA0160)
The Acetylcholinesterase Inhibition Assay Kit (SKU: BA0160) provides a rapid colorimetric method for screening acetylcholinesterase (AChE) inhibitors. Acetylcholinesterase catalyses the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid, a reaction necessary to allow a cholinergic neuron to return to its resting state, and its inhibition is an important target for the management of Alzheimer's disease. This assay is based on an improved Ellman method, in which thiocholine produced by the action of acetylcholinesterase forms a yellow colour with 5,5'-dithiobis(2-nitrobenzoic acid). The intensity of the product colour, measured at 412 nm, is proportional to the enzyme activity in the sample. The homogeneous mix-incubate-measure format can be completed in under 30 minutes and is readily automated for high-throughput screening.
| Product Name: | Acetylcholinesterase Inhibition Assay Kit (Colorimetric) (BA0160) |
| SKU: | BA0160 |
| Detection Method: | Colorimetric |
| Detection Range: | Percent inhibition (screening assay) |
| Sample Type: | Purified acetylcholinesterase and test compounds |
| Species Reactivity: | All |
| Assay Time: | Under 30 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store the substrate and DTNB at -20 C and all other components at room temperature upon receipt. |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
A rapid colorimetric screening assay for acetylcholinesterase inhibitors based on an improved Ellman method. Thiocholine produced by acetylcholinesterase reacts with DTNB to form a yellow product measured at 412 nm, with intensity proportional to enzyme activity; inhibitors reduce this signal. The assay is homogeneous and readily automated. Neither acetylcholinesterase nor a control inhibitor is supplied with the kit.
- Rapid and reliable, completed in under 30 minutes
- High-throughput homogeneous mix-incubate-measure format
- Based on an improved Ellman method
- Readily automated on HTS liquid-handling systems
- High-throughput screening and evaluation of acetylcholinesterase inhibitors
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 | Prepare samples: dilute purified AChE to 400 U/L using assay buffer and dissolve test compounds in a suitable solvent, verifying enzyme tolerance if using DMSO. |
| 2 | Equilibrate all components to the desired reaction temperature and prepare the Working Reagent freshly for use within 30 min. |
| 3 | Transfer 45 uL AChE into separate wells and 45 uL assay buffer into a No Enzyme Control well. Add 5 uL solvent to the No Enzyme Control and one No Inhibitor Control well, and 5 uL of each test compound to the remaining AChE wells. Incubate the plate for 15 min. |
| 4 | Prepare Working Reagent per well by mixing 154 uL Assay Buffer, 1 uL Substrate and 0.5 uL DTNB. Add 150 uL to each sample, sample blank and No Inhibitor Control well and tap to mix. |
| 5 | Read optical density at 412 nm at 0 min and at 10 min. |
| 6 | A 384-well format is also supported using 18 uL AChE, 2 uL inhibitor and 30 uL Working Reagent. |
Calculate percent inhibition = (1 - (dOD Test Compound / dOD No Inhibitor)) x 100%, where dOD Test Compound is the change in OD at 412 nm between 0 and 10 min for a test compound well and dOD No Inhibitor is the corresponding change for the No Inhibitor Control well.
| Component | Quantity | Storage |
| Assay Buffer (pH 7.5) | 30 mL | Room temperature |
| DTNB | 60 uL | -20 C |
| Substrate (100 mM) | 500 uL | -20 C |
