2010-09 ache assay Protocol
1. Compound and equipment: 1) Phosphate: 0.1 M, ph 8.0. ①Begin by making the following volumes of the specified solutions. Volume (A) 0.2M sodium phosphate monobasic ②Weigh 0.552g nah2po4 and add to 20ml of distilled H2O Volume (B) 0.2M sodium phosphate dibasic ③Weigh 2.83g Na2HPO4 and add to 100ml of distilled H2O Combine the two solutions according to the following: Vol (A) = 5.3ml Vol (B) = 94.7ml ④The resulting solution will have a final concentration of 0.2M, [ph=8] and will yield 100ml. ⑤Dilute buffer by adding an equal volume of distilled H2O ~ 100ml. The working concentration of the new solution will be 0.1M phosphate buffer, ph=8. ⑥Filter sterilize with 1L filter system and autoclave for 25min.
2) Substrate: Acetylthiocholine iodide, 0.075 M (21.67 mg/ml). This solution was used successfully for l0-15 days if kept refrigerated. 3) Reagent.: Dithiobisnitrobenzoic acid (DTNB): 0.01 M of the 5’ 5-dithiobis-2- nitrobenzoic acid , 39.6 mg were dissolved in 10 ml ph 7.0 phosphate buffer (0.1 M) and 15 mg of sodium bicarbonate were added. The reagent was made up in buffer of ph 7 in which it was more stable than in that of ph 8. 4) ???Enzyme: Bovine erythrocyte cholinesterase (Nutritional Biochem. Corp., 20,000 units) was dissolved in 20 ml of 1% gelatin. This solution was diluted 1 ：200 with water for use, yielding a solution of 5 units/ml.⇒ maybe for blood samples not for brain tissues 5) Ethopropazine hydrochloride: to inhibit nonspecific cholinesterase activity, added to the incubation mixture or the assay mixture. For brain ache, butyryl cholinesterase was inhibited by the addition of 10μm ethopropazine to the assay mixture and the change in absorbance was measured at 412 nm for 2min at 30 s intervals.
6) Versa Max Microplate Reader: (Molecular Devices Corp., Sunnyvale, CA).
7) Pipeting (multi-channel) devices. Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader.
Price of compound
1) Ethopropazine hydrochloride ≥98% (HPLC).2. 2) A 0.0.000 KRW D8130 Sigma
. 0. 20 μ1 were added.
1) The tissue was homogenized (approximately 20 mg of tissue per ml of phosphate buffer (ph 8.5′-Dithiobis(2-nitrobenzoic acid) ≥97.0. 5) The rates were calculated as follows:
3.6 ml of phosphate buffer (ph 8.000 KRW D218200-5G 113.000 KRW E5406-250MG 832.000 KRW
2) 5. 4) Of the substrate.1 M). the photometer slit was opened so that the absorbance was set to zero. The absorbance was measured at 412 nm. powder
E5406 Sigma E5406-50MG 185.000 KRW D218200 Aldrich D218200-1G 44. Changes in absorbance were recorded and the change in absorbance per min.1 M) by a pestle (Scienceware. Pequannock. 100 μ1 were added to the photocell.4-ml aliquot of this homogenate was added to a cuvette containing 2. USA) (For muscular tissue.5% (HPLC)
43760 Fluka 43760-1G 39. when this had stopped increasing. considerable mincing was necessary before homogenizing).000 KRW 43760-25G 356. Was calculated. 3) Of the DTNB reagent.000 KRW D218200-10G 223.
D8130-500MG 23.000 KRW D8130-10G 218. Detection limit: 10 U/L. Shipping: ambient temp. powder
A5751 Sigma A5751-250MG 43. storage: room temp.000 KRW D8130-1G 58.000 KRW
3) Acetylthiocholine iodide ≥98% (TLC).000 01480-25G 345. Procedure: 10 min. 5) Amplite™ Colorimetric Acetylcholinesterase Assay Kit $195 USD Product Number: #11400 (200 assays) Keep in freezer and avoid light Absorbance microplate readers
4) Quantichrom™ Acetylcholinesterase Assay Kit( DACE-100)
$299.000 A5751-5G 111.00 USD Quantitative determination of acetylcholinesterase activity by colorimetric (412nm) method.000 A5751-25G 343.000 KRW D8130-25G 516. Shelf life: 6 months.000 A5751-1G 49.000 KRW D8130-5G 118.000
01480 Fluka 01480-1G 34.000 01480-5G 88. Kit size: 100 tests.
PROCEDURES Samplepreparation. High-throughput. a reaction necessary to allow a cholinergic neuron to return to its resting state after activation. Evaluation of acetylcholinesterase inhibitors. Detection range 10 to 600 U/L ache activity in 96-well plate assay.000 rpm for 5 min. KEY FEATURES Sensitive and accurate. Please refer to Material Safety Data Sheet for detailed information. Ache catalyzes the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid. Can be readily automated as a high-throughput 96.7. measured at 412 nm. APPLICATIONS Direct assays of acetylcholinesterase activity in blood. in which thiocholine produced by the action of acetylcholinesterase forms a yellow color with 5. followed by centrifugation at 14. direct and automation-ready procedures for measuring ache activity are very desirable. ache). The intensity of the product color. serum. plasma.1.well plate assay for thousands of samples per day. Use
. and other biological samples.Quantichrom™ Acetylcholinesterase Assay Kit( DACE-100) protocol
DESCRIPTION ACETYLCHOLINESTERASE (EC 3. Cholinesterase levels of cells and plasma are used as a guide in establishing safety precautions relative to exposure and contact.5’-dithiobis(2-nitrobenzoic acid). Normal precautions for laboratory reagents should be exercised while using the reagents. also known as RBC cholinesterase.1M phosphate buffer (ph 7. is proportionate to the enzyme activity in the sample.5). is found primarily in the blood and neural synapses. as well as a guide in determining the need for workers to be removed from areas of contact with the organic phosphate insecticides. Tissue or cell lysates are prepared by brief sonication or homogenization in 0. Precautions: reagents are for research use only. Low serum cholinesterase activity may relate to exposure to insecticides or to one of a number of variant genotypes. The procedure involves adding a single working reagent. and reading the optical density at 2 min and 10 min at room temperature. Simple. Quantichromtm Acetylcholinesterase Assay is based on an improved Ellman method.1.
Reaction: transfer 190 µl freshly prepared Working Reagent to all sample wells and tap plate briefly to mix. Calculation: acetylcholinesterase activity is calculated as follows. Ache Activity = (OD10 – OD2 / OD CAL – ODH2o ) × n × 200 (U/L)
Where OD10 and OD2 are the OD412nm values of the sample at 10 min and 2 min. Calibrator: transfer 200 µl water and 200 µl calibrator separately into wells of a clear bottom 96-well plate. dilute sample in Assay Buffer and repeat this assay.supernatant for assay.5 and room temperature). 3.respectively. Samples: add 10 µl sample per well in separate wells. 1. Reagent preparation: the Working Reagent should be prepared freshly and used within 30 min. Add 200 µl Assay Buffer per 2 mg reagent. ODCAL and ODH2O are the OD412nm values of the Calibrator and water at 10 min. Ideally samples should be assayed fresh. 2. Calculate the amount of reagent needed and weigh this amount (mg) in a centrifuge tube. Note: if the calculated ache activity is higher than 600 U/L.
. The number “200” is the equivalent activity of the calibrator under the assay conditions. Each reaction well requires 2 mg reagent. Vortex to dissolve. N is the dilution factor (n = 40 for whole blood). If this is not possible. refrigerate samples and assay them within 24 hours. Unit definition: one unit of enzyme catalyzes the production of 1 µmole of thiocholine per minute under the assay conditions (ph 7. Multiply the results by the dilution factor.