Invitrogen™ Amplex™ Acetylcholine/Acetlycholinesterase Assay Kit

Catalog No.	Quantity
A12217	500 Assays

Catalog No. A12217 Supplier Invitrogen™ Supplier No. A12217

Description

Detect activity levels as low as 0.002U/mL in one hour

The Amplex™ Red Acetylcholine/Acetylcholinesterase Assay Kit provides an ultrasensitive method for detecting acetylcholinesterase (AChE) activity in a fluorescence microplate reader or fluorometer.

• Detect AChE activity levels as low as 0.002 U/mL in one hour
• Detect acetylcholine levels as low as 0.3 μM using excess AChE
• Achieve detection ranges of 0.3 μM to 100 μM acetylcholine
• Format allows for multiple time point measurements
• Designed for minimal autofluorescence interference

AChE activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex™ Red reagent), a sensitive fluorogenic probe for hydrogen peroxide. AChE converts the acetylcholine substrate to choline, which is then oxidized by choline oxidase to betaine and hydrogen peroxide. In the presence of horseradish peroxidase, hydrogen peroxide reacts with the Amplex™ Red reagent in a 1:1 stoichiometric ratio to generate the highly fluorescent product resorufin.

Because resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, there is little interference from autofluorescence in most biological samples. Experiments with purified AChE from electric eel indicate that the Amplex™ Red Acetylcholine/Acetylcholinesterase Assay Kit can detect AChE levels as low as 0.002 U/mL using a reaction time of one hour. By providing an excess of AChE in the assay, the kit can also be used to detect acetylcholine levels as low as 0.3 μM, with a range of detection from 0.3 μM to 100 μM acetylcholine.

Use Amplex™ Red Assays for a Broad Range of Investigations

A wide variety of validated Amplex™ Red assays are available for studying cell signaling and lipids, neurobiology, inflammation and immune function, and metabolism. We also offer Amplex™ UltraRed Reagent (Cat. No. A36006), a second-generation reagent providing greater sensitivity and brighter fluorescence, and the Amplex™ Red/UltraRed Stop Reagent (Cat. No. A33855). The Amplex™ Red/UltraRed Stop Reagent provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After addition of the stop reagent, the fluorescence signal remains stable for at least three hours. Custom assay design and packaging are also available.

Order Info

Shipping Condition: Room temperature

Specifications

• Content And Storage: Store in freezer -5°C to -30°C and protect from light.
• Detection Method: Fluorescence
• For Use With (Application): Enzyme and Metabolic Activity
• For Use With (Equipment): Microplate Reader
• Product Type: Amplex Red Assay Kit
• Dye Type: Amplex Red
• Product Line: Amplex
• Quantity: 500 Assays
• Shipping Condition: Room Temperature
• Substrate Properties: Molecular Substrate
• Substrate Type: HRP (Horseradish Peroxidase) Substrate, Acetylcholinesterase Substrate
• Target Enzyme: Acetylcholinesterase
• Excitation Wavelength: 571 nm
• Emission Wavelength: 585 nm

Frequently Asked Questions (FAQs)

I'm using an Amplex Red kit, the reagent changes color to pink almost immediately in my own Krebs-Ringer buffer but not in HBSS. Why is this?

The components of Krebs-Ringer buffer (salts) should not cause oxidation of the Amplex reagent (which, in the presence of peroxidase and H₂O₂ oxidizes to resorufin, which is pink in color and fluorescent). Try water alone (the water used to make the Krebs-Ringer buffer). Since Hank's Buffered Saline Solution is typically purchased rather than made in the lab, it likely would not have the same contaminant. Another option is to degas the buffer prior to use to removed dissolved oxygen radicals.

Can Amplex Red Assays be performed using cell lysates?

This is not recommended. The presence of endogenous proteases can complicate the assay by degrading the horseradish peroxidase (HRP). Endogenous peroxidases and antioxidants can modify the H₂O₂ required for the reaction, competing with HRP (and catalase) for the substrate.

The Amplex Red Assays are best performed with either purified enzymes or extracted H₂O₂ in a defined buffer system, extracellular solutions or body fluids (media, serum, etc.) that do not exhibit high levels of endogenous protease or oxidase activity and do not contain antioxidants.

For Research Use Only. Not for use in diagnostic procedures.