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Caspase / Apoptosis Detection

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Mitochondria Membrane Potential detection

	Mitochondria Membrane Potential - JC 1
	MM Potential detection for Flow Cytometry - Mito Flo

aCella - Bioluminescence Assay Kits

	Non Radioactive Assay for Cytotoxicity - aCella TOX
	Monitoring Acetylcholinesterase activity - aCella AChE New!!!

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Cathepsin B,K,L Detection Kits

	Substrate based Assay to detect protease activity in hole living cells

aCella™ - AChE
Bioluminescence Non Radioactive Assay for monitoring Acetylcholinesterase activity * Patent Pending

Key Benefits:

Safe - Non Radioactive Enzyme activity assay.
Versatile: Nerve Gas, Pesticide Monitoring, drug screening Applications
Homogenous - One-step, no wash assay.
FAST - Results in 30 secs - 5 minutes.
Highly Sensitive
HTS - Adaptable for High Throughput format
Applications - Standard luminometer readout

Introduction to aCella - AChE

Acetylcholinesterase (AChE) is one of the most important enzymes involved in nerve transmission. The enzyme is bound to cellular membranes of excitable tissue (synaptic junction, endoplasmic reticulum, etc)^1-3. Acute toxicity to humans and animals through inhibition of AChE by both nerve gases and an important class of pesticides has long been a field of intensive scientific investigation ^4,5. AChE inhibitors have also been used clinically as Alzheimer’s treatments (e.g., tacrine (tetrahydroaminoacridine)) ⁽⁶⁾ and are the subject of increasing interest in various disease processes and treatment strategies ^7,8. However, both environmental detection of AChE inhibitors and development of modulators of AChE enzymatic activity as drugs have been hampered by the difficulty and complexity of the current assay methods.

Assay Principle:

We have developed a highly sensitive, very rapid, extremely simple assay for AChE activity, using the natural substrate, acetylcholine. As shown in Figure 1, a series of coupled enzyme reactions quickly translates the presence of active AChE into a change in the luminance of the reaction. First (reaction I), acetylcholine is hydrolyzed by the AChE to yield acetate and choline. The acetate and choline then enter a coupled enzyme reaction (reaction II) that results in consumption of ATP, and finally the ATP concentration is measured by the well-established luciferase method (reaction III). These reactions can occur simultaneously, and the result is generally obtained in five minutes or less. Inhibitors of AChE are readily detected by an increase in luminance due to reduced consumption of ATP.

The following reaction illustrates the sequence of events if AChE inhibitors are present:

Reaction I: AChE + Inhibitor No Acetate and Choline

Reaction II: Coupled Enzyme Reaction + ATPReaction does not proceed

Reaction III: ATP (remaining) + Luciferase/LuciferinLIGHT

References:

Politoff, A., Blitz, A., and Rose, S.: Incorporation of Acetylcholinesterase Into Synaptic Vesicles is Associated with Blockade of Synaptic Transmission, Nature 256, 324, 1975
Friedenberg, R., and Seligman, A.: Acetylcholinesterase at the Myoneural Junction: Cytochemical Ultrastructure and Some Biochemical Considerations,
J Histochem Cytochem 20, 771, 1972
Nachmansohn, D.: Proteins in Excitable Membranes, Science 168, 1059, 1970.
(HA Berman and MM Decker. Kinetic, equilibrium, and spectroscopic studies on dealkylation ("aging") of alkyl organophosphonyl acetylcholinesterase. Electrostatic control of enzyme topography. J. Biol. Chem., Aug 1986; 261: 10646-10652 .
Arie Ordentlich et al. The Architecture of Human Acetylcholinesterase Active Center Probed by Interactions with Selected Organophosphate Inhibitors. J. Biol. Chem., May 1996; 271: 11953-11962.
Levy R. Tetrahydroaminoacridine and Alzheimer's disease. Lancet, 1987 Feb 7;1(8528):322.
Bolognesi ML et al. Propidium-based polyamine ligands as potent inhibitors of acetylcholinesterase and acetylcholinesterase-induced amyloid-beta aggregation. J Med Chem. 2005 Jan 13;48(1):24-7.
Schallreuter KU et al. Activation/deactivation of acetylcholinesterase by H₂0₂: more evidence for oxidative stress in vitiligo. Biochem Biophys Res Commun. 2004 Mar 5;315(2):502-8.

Kit Content:

Component A: Contains Acetylcholinesterase ............Part# 3023
Component B: Contains Detection reagent, acetylcholine and kinase enzymes..Part# 3024
Component C: Control to measure maximum Luminescence.....Part# 3025

The following kits are available:
Catalog #	Size*	Price
CLACHE 100-2	100	$395
CLACHE 100-3	500	$1295
CLACHE 100-4	1000	$2195

* Please call 888 7 ASSAYS (888-727-7297) or email info@celltechnology.com for volume pricing

Product Data Sheet

Protocol

MSDS

Figure 1. Tacrine ( a mixed-mode inhibitor of AChE) was serially diluted in DI water. Next 10uL of the diluted Tacrine (x axis labeling represents uM final concentration of Tacrine) was added to a white opaque 96 well microplate along with 50 uL of component A (AChE enzyme). The samples were incubated for 5 minutes after which 50uL of component B was added to all the wells. Data were collected using a luminometer. Data shown represents T=2 minutes after the addition of component B.

Figure 2. Malathion, a common pesticide, was first diluted in DMSO and subsequently serially diluted in Di water. 10uL of the diluted Malathion (x axis represents uM final concentration of Malathion) was added to a white opaque 96 well microplate followed by 50 uL of component A (AChE enzyme). The mixture was incubated for 15 minutes, after which 50 uL of component B was added to all the wells. Data were collected using a luminometer. Data shown represents T=2.5 minutes after the addition of component B.

Optimized with

Veritas Luminometers from Turner Biosystems