Log In/Register
Acetylcholinesterase Inhibitor Screening Service
Summary
In order to develop an Acetylcholinesterase inhibitor screening assay kit, we performed some initial experiments to establish appropriate assay conditions. We assessed conditions for Acetylcholinesterase from electric eel. First, we titrated the AChE using 1 mM Acetylthiocholine iodide as the substrate and DTNB as the chromagen. From this titration, we decided to use 0.0035 Units/reaction AChE for assay development and IC50 determinations. The Km of Acetylthiocholine iodide for AChE was then determined. We next titrated DMSO to determine the highest concentration that would be tolerated by AChE. Finally, the IC50’s for two known Acetylcholinesterase inhibitors, Donepezil and Physostigmine, were measured. We determined the Km to be 0.425 mM and the maximum DMSO concentration to be 100 v%. Using 0.425 mM acetylthiocholine iodide, we observed an IC50 = 48.3 nM for Donepezil and an IC50 = 46.7 nM for Physostigmine when each inhibitor was dissolved in 20v% DMSO.
Results
AChE Titration
In order to determine an appropriate amount of AChE to use for the inhibitor screening assay and for IC50 determinations, we titrated AChE. The reaction was performed using 1 mM Acetylthiocholine Iodide and 0.4 mM DTNB dissolved in Assay Buffer pH 7.6. From these results, we opted to use 0.0035 U/rxn AChE. At this concentration, the reaction would remain linear for at least 10 minutes, and the ΔOD should be significant (>0.2) even at the Acetylthiocholine Iodide Km.
Km of Acetylthiocholine Iodide
In order to determine the Km of Acetylthiocholine iodide for Acetylcholinesterase (electric eel), we titrated acetylthiocholine iodide from 0-4.3 mM with 0.0035 U/rxn AChE and computed ΔOD (OD10min – OD0min), which is proportional to the rate of reaction, for acetylthiocholine iodide. The ΔOD’s were then plotted versus acetylthiocholine iodide concentration and the Km was computed using Prism 4 (GraphPad Software Inc.). We found the Km = 0.425 mM for Acetylthiocholine Iodide.
DMSO Titration
In order to determine tolerance for DMSO of Acetylcholinesterase, we ran a DMSO titration (from 0-100 v% for the 5 µL of variable compound in a 200 µL reaction). For this titration we used 0.0035 U/rxn AChE. The reaction was initiated by adding Acetylthiocholine Iodide, DTNB, and Assay buffer pH 7.6 (Final concentrations 0.425 mM Acetylthiocholine Iodide and 0.3 mM DTNB) and was allowed to proceed for 10 min. We then computed the ΔOD (OD10min – OD0min), which is proportional to the rate of reaction, for each DMSO concentration. The ΔOD’s were then plotted versus DMSO concentration. From these results, we discovered that that we can use up to 100 v% DMSO if needed, but decided to move forward with 20 v% DMSO for IC50 determinations.
Donepezil IC50 Determination
The IC50 determination required two steps: 1) 15 min pre-incubation of 45 µL AChE (78 U/L) with 5 µL Donepezil (in 20 v% DMSO), and 2) acetylcholinesterase reaction with 150 µL Working reagent containing acetylthiocholine iodide, DTNB, and assay buffer for 10 min (0.425 mM [Acetylthiocholine Iodide] and 0.31 mM [DTNB] final). We titrated Donepezil from 0-25 µM and computed the ΔOD for each Donepezil concentration. The ΔOD’s were then plotted versus the Log [Donepezil] and the IC50 was computed using Prism 4 (GraphPad Software Inc.). We observed an IC50 = 48.3 nM.
Physostigmine IC50 Determination
The IC50 determination required two steps: 1) 15 min pre-incubation of 45 µL AChE (78 U/L) with 5 µL Physostigmine (in 20 v% DMSO), and 2) acetylcholinesterase reaction with 150 µL Working reagent containing acetylthiocholine iodide, DTNB, and assay buffer for 10 min (0.425 mM [Acetylthiocholine Iodide] and 0.31 mM [DTNB] final). We titrated Physostigmine from 0-25 µM and computed the ΔOD for each Physostigmine concentration. The ΔOD’s were then plotted versus the Log [Physostigmine] and the IC50 was computed using Prism 4 (GraphPad Software Inc.). We observed an IC50 = 46.7 nM.
Please email or call 1-510-782-9988 x 2 to request assay service.
Customer Testimonials
"The guys at BioAssay Systems know their science! I had to develop a bioassay for our company and Robert was very quick to respond to keep us up to date. They are knowledgeable and very resourceful. They directed us to one of the bulk suppliers that could give us a really good deal on our active ingredient. They keep very detailed records for our research needs. I would recommend working with BioAssay Systems on your assay development projects!"
"It is a pleasure working together with BioAssay Systems. I brought samples to their Hayward location and received the results in less than 24 hr. The discussion with Robert Z was courteous, open, and with a depth of knowledge. Robert has a PhD from Stanford. Also, Frank Huang, the CEO, had sufficient scientific curiosity to try out our cleanser and provide feedback! This is beyond the call of customer relations. BioAssay Systems is a company with a lot of expertise and a friendly, responsive approach to problem solving. I will enthusiastically continue to work with them to carry out our research."
"I would like to show my appreciation to the Bioassay Systems service team. We are developing an assay to measure the change in lactate concentration of single cell samples using fluorescent microscopy. The BioAssay Systems team was quite helpful in assisting us to optimize their fluorescent L-Lactate assay kit for our unique experimental set-up. The BioAssay Systems team is always friendly and knowledgeable. We know that we can rely on them for fast and professional service."
"While developing biodegradable coatings for Drug Eluting Stents I worked with Bob to develop an accurate, precise and sensitive assay for determining the amount of polymer remaining in tissue. It was a pleasure to work with Bob and his team, they are very thorough, they understand the science very well and they always deliver on time and at the right price. I would highly recommend anyone using BioAssay Systems services in the future."
Previous
Next
