Acetylcholinesterase Fluorescent Activity Kit, 2 Plate

N° du produit ABIN577658

Détail du produit

Antigène: Acetylcholinesterase (AChE)

Reactivité: Rat, Humain, Souris, Poulet, Chien, Singe, Porc

Méthode de détection: Fluorometric

Seuil minimal de détection: 0.321 mU/mL

Application: Activity Assay (AcA)

Fonction: The DetectX® Acetylcholinesterase Activity kit is designed to quantitatively measure acetylcho-linesterase (AChE) activity in a variety of samples.

Marque: DetectX®

Type d'échantillon: Serum, Plasma, Erythrocyte Membranes

Analytical Method: Quantitative

Specificité: Species Independent. Samples Types validated: Serum, Plasma, and Erythrocyte Membranes

Réactivité croisée (Details): A sample of native human butyrylcholinesterase at 20 mU/mL was tested in the assay. It read 2.45 times higher than the same concentration of AChE tested at the same time.

Sensibilité: 0.218 mU/mL

Attributs du produit: The Acetylcholinesterase (AChE) Activity kit is designed to quantitatively measure AChE activity in a variety of samples. A human AChE standard is provided to generate a standard curve for the assay. The kit utilizes a proprietary non-fluorescent molecule, ThioStar®, that covalently binds to the thiol product of the reaction between the AChE Substrate and AChE in the standards or samples, yielding a fluorescent product read at 510 nm in a fluorescent plate reader with excitation at 390 nm. Acetylcholinesterases (AChE) appear critical to both development and function of the nervous system. The use of AChE inhibitors as therapeutic agents and pesticides has spurred detailed investigations of cholinesterases since their identification by Dale. Acetylcholine (ACh) is an essential neurotransmitter in the central and peripheral nervous systems. In the brain multiple areas exist where cholinergic neurons are concentrated. Nicotinic and muscarinic ACh receptors are recognized as binding and effector proteins to mediate chemical neurotransmission at neurons, ganglia, heart and smooth muscle fibers and glands. This traditional view of AChE acting solely as neurotransmitter has to be revised based on the findings published both early and late in the last century, demonstrating the non-neuronal cholinergic system.

Ingrédients: Black 96 Well Plates 2 Plates
Acetylcholinesterase Standard 225 μL Acetylcholinesterase (AChE) at 1,000 mU/mL in a special stabilizing solution. CAUTION: This material is isolated from human erythrocytes. Treat as potentially infectious.
ThioStar® Detection Reagent 2 vials ThioStar thiol detection substrate stored in a desiccator. Reconstitute with dry DMSO.
Dry DMSO 14 mL Dry Dimethyl sulfoxide solvent over molecular sieves. May be stored at room temperature.
Assay Buffer Concentrate 28 mL A 10x concentrated Tris buffer containing detergents and stabilizers.
AChE Substrate 2 vials Acetylthiocholine iodide freeze dried with stabilizers.

Matériel non inclus: Distilled or deionized water.
Repeater pipet with disposable tips capable of dispensing 50 μL.
Fluorescence 96 well plate reader capable of reading fluorescent emission at 510 nm, with excita- tion at 390 nm.
Contact your plate reader manufacturer for correct filter sets.
Set plate param- eters for a 96-well Corning Costar 3915 plate.
See: http://www.arborassays.com/resources/ Software for converting raw relative fluorescent unit (FLU) readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting.

Information d'application

Indications d'application: This assay has been validated for serum, EDTA and heparin plasma, and solubilized RBC ghosts from a variety of species.
Samples containing visible particulate should be centrifuged prior to using.

Commentaires:

Sample values: A variety of serum and plasma samples were tested in the assay, including chicken, mouse, rat, dog, monkey, pig and human samples.
Values averaged 19,188 mU/mL.
RBC ghost samples ranged from 6,216 to 18,552 mU/mL with an average of 13,158 mU/mL

Durée du test: 1 h

Plaque: Uncoated

Protocole: A human AChE standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve.
The kit utilizes a proprietary non- fluorescent molecule, ThioStar®, that covalently binds to the thiol product of the reaction between the AChE Substrate and AChE in the standards or samples, yielding a fluorescent product read at 510 nm in a fluorescent plate reader with excitation at 390 nm.
The kit is suitable for measuring AchE activity in appropriately diluted serum, plasma and RBC ghosts from a number of species.
It will also measure AChE in extracted tissue samples and cell lysates.
Because the readout of AChE activity is purely chemical, there are few interferants that will affect the readings obtained.
REACTION OVERVIEW
1.
Sample or standard added to well.
2.
The reaction is initiated with the addition of the Reaction Mix containing AChE Substrate and ThioStar® Reagent.
3.
Incubate for 20 minutes and read fluorescent signal.
Calculate AChE activity from standard curve.
4.
Alternatively samples can be read kinetically.
Follow steps 1 and 2 above.
Add Reaction Mix and read signal at 510 nm over time.
Compare rates for samples and standards to determine sample AChE activity.

Préparation des réactifs:

Allow the kit reagents to come to room temperature for 30 minutes.
We recommend that all stan- dards and samples be run in duplicate to allow the end user to accurately determine AChE activ- ity.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Assay Buffer Prepare the Assay Buffer by diluting one part of the 10x Assay Buffer Concentrate with nine parts deionized water for a 1:10 dilution.
It is stable for up to 3 months when stored at 4 °C. ® www.ArborAssays.com 7 WEB INSERT 150728 reagent preparatiOn cOntinued ThioStar® Detection Reagent Allow the desiccator to warm to room temperature prior to opening.
Remove a vial of ThioStar Reagent from the desiccator and add 700 μL of the provided DMSO to the vial and vortex thor- oughly.
Store any unused reconstituted Detection Reagent at 4 °C in the desiccator and use within 2 months.
Acetylcholinesterase Substrate Add 700 μL of the provided DMSO to the AChE Substrate vial and vortex thoroughly.
This is a 10x concentrate of the substrate.
Store any unused reconstituted AChE Substrate at room tempera- ture and use within 2 months.
Reaction Mix Dilution Table 1/2 Plate Full Plate 10X AChE Substrate Concentrate 300 μL 550 μL 10X ThioStar® Concentrate 300 μL 550 μL DMSO 2.4 mL 4.4 mL Standard Preparation AChE Standards are prepared by labeling five test tubes as #1 through #5.
Briefly spin vial of stan- dard in a microcentrifuge to ensure contents are at bottom of vial.
Pipet 450 μL of Assay Buffer into tube #1 and 250 μL into tubes #2 to #5.
Carefully add 50 μL of the AChE Standard to tube #1 and vortex completely.
Take 250 μL of the AChE solution in tube #1 and add it to tube #2 and vortex completely.
Repeat these serial dilutions for tubes #3 through #5.
The activity of AChE in tubes 1 through 5 will be 100, 50, 25, 12.5, and 6.25 mU/mL.
Use all Standards within 2 hours of preparation.

Préparation de l'échantillon:

Serum & Plasma Store separated serum or plasma on ice until assaying or freeze in aliquots for later use. Samples must be diluted in Assay Buffer prior to running in the kit. Any samples with AChE activity outside the standard curve range should be diluted further with Assay Buffer to obtain readings within the standard curve. Serum and plasma typically have to be diluted ≥ 1:300 to read in the assay. Erythrocytes (RBCs) Blood is collected in the presence of heparin or EDTA. The sample is then centrifuged and the plasma and white cell layer are removed from the RBC layer. The RBCs are suspended and gently washed twice with three volumes of isotonic saline (0.9 % ), separating the cells by centrifugation at 600 x g for 10 minutes and discarding the saline after each step. To lyse the RBCs, four volumes of cold deionized water are added to the RBCs. The cells are then vortexed and incubated for 10 minutes at 4°C, or allowed to undergo a freeze/thaw. Samples are centrifuged at 14,000 rpm for 10 minutes at 4°C and the supernatant discarded. Further wash the membrane pellet with two or three volumes of isotonic saline, with centrifuga- tion in between, until it is only slightly pink. The smaller dark red pellet on the bottom is non-lysed RBCs and should be avoided. Solubilize the white membrane ghost pellet with Triton X-100. Final assay dilution of the solubilized RBC ghost sample must be sufficient that the assay sample con- tains ≤ 0.01 % Triton X-100. Use all samples within 2 hours of dilution.

Procédure de l'essai:

1. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification. Set plate parameters for a 96-well Corning Costar 3915 plate. See: http://www.ArborAssays.com/resources/lit.asp for plate dimension data.
   2. Pipet 100 μL of samples or standards into duplicate wells in the plate.
   3. Pipet 100 μL of Assay Buffer into duplicate wells as a Zero standard.
   4. Add 50 μL of the prepared Reaction Mix to each of the wells using a repeater pipet.
   5. Gently tap the sides of the plate to ensure adequate mixing of the reagents.
   6. Incubate at room temperature for 20 minutes.
   7. Read the fluorescent emission at 510 nm with excitation at 370-410 nm. Please contact your plate reader manufacturer for suitable filter sets.

Calcul des résultats:

Average the duplicate FLU readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean FLUs for the zero standard.
The sample activity obtained should be multiplied by the dilution fac- tor to obtain neat sample values.
Or use the online tool from http://www.myassays.com/arbor-assays-acetylcholinesterase-fluorescent-activity-kit.assay to calculate the data. The MyAssays logo is a registered trademark of MyAssays Ltd.

Restrictions: For Research Use only

Stockage

Précaution d'utilisation: As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
Dimethyl sulfoxide is a powerful aprotic organic solvent that has been shown to enhance the rate of skin absorption of skin-permeable substances.
Wear protective gloves when using the solvent especially when it contains dissolved chemicals.
The Acetylcholinesterase Standard is derived from human blood.
It has been extensively tested for viral contamination, but all human blood products should be treated as potentially infectious and adequate precautions taken.
ThioStar® Detection Reagent should be stored at 4°C in the desiccator.
Allow to warm to room temperature prior to opening.
ThioStar will react with strong nucleophiles.
Buffers containing the preservatives sodium azide, Proclin™ and Kathon™ will react with the substrate.
Reconstituted ThioStar in DMSO stored at 4°C in the supplied desiccator can be used up to 2 months later.
The background on the reconstituted ThioStar will increase slowly over time but the increase will not affect the assay results obtained.

Stock: 4 °C,RT

Stockage commentaire: All components of this kit should be stored at 4°C until the expiration date of the kit. DMSO, when stored at 4°C, will freeze. Can be stored tightly capped at room temperature.

Référence

Yusuf, Khan, Khan, Ahmed: "Preparation, characterization, in vivo and biochemical evaluation of brain targeted Piperine solid lipid nanoparticles in an experimentally induced Alzheimer's disease model." dans: Journal of drug targeting, (2012) (PubMed).

Détail du antigène

Antigène: Acetylcholinesterase (AChE)

Autre désignation: Acetylcholinesterase (AChE Produits)

Synonymes: ACEE Kit, ARACHE Kit, N-ACHE Kit, YT Kit, Ache Kit, GB14873 Kit, zgc:92550 Kit, ACE Kit, Dsim\\GD20515 Kit, GD20515 Kit, dsim_GLEANR_4292 Kit, ache Kit, mE1a Kit, mE1b Kit, mE1c Kit, mE1c-long Kit, mE1d Kit, mE1d' Kit, mE1e Kit, arache Kit, n-ache Kit, acetylcholinesterase (Cartwright blood group) Kit, acetylcholinesterase 2 Kit, acetylcholinesterase Kit, Acetylcholine esterase Kit, acetylcholinesterase (Cartwright blood group) L homeolog Kit, collagen type I alpha 2 chain Kit, ACHE Kit, AChE-2 Kit, Ache Kit, ache Kit, Dsim\Ace Kit, ache.L Kit, COL1A2 Kit, ACE-1 Kit

Sujet: Acetylcholinesterases (AChE) appear critical to both development and function of the nervous system. The use of AChE inhibitors as therapeutic agents and pesticides has spurred detailed investigations of cholinesterases since their identification by Dale1. Acetylcholine (ACh) is an es- sential neurotransmitter in the central and peripheral nervous systems. In the brain multiple areas exist where cholinergic neurons are concentrated2. Nicotinic and muscarinic ACh receptors are recognized as binding and effector proteins to mediate chemical neurotransmission at neurons, ganglia, heart and smooth muscle fibers and glands. This traditional view of AChE acting solely as neurotransmitter has to be revised based on the findings published both early and late in the last century, demonstrating the non-neuronal cholinergic system. O N+ + HO NO Choline Acetylcholine Acetylcholinesterase is encoded by the single AChE gene, and the structural diversity in the gene products arises from alternative nRNA splicing and post translational associations of catalytic and structural subunits. The major form of acetylcholinesterase found in brain, muscle, and other tissues is the hydrophilic species, which forms disulfide-linked oligomers with collagenous, lipid- containing structural subunits. The other alternatively-spliced form, expressed primarily in the erythroid tissues, is structurally different at the C-terminal end and contains a cleavable peptide with a GPI anchor. It associates with membrane receptors through phosphoinositide moieties added post-translationally3. Impairment of cholinergic neurotransmission is well-established in Alzheimer's disease, but there is controversy about its relevance at the early stages of the disease as well as in mild cognitive impairment. In vivo positron emission tomography imaging of corti- cal AChE activity as a marker of cholinergic function that is expressed by cholinergic axons and neurons has demonstrated a reduction of this enzyme activity in manifest Alzheimer's patients4. Other intentional or environmental methods of impairment is with organophosphates and car- bamates with anticholinergic properties which are used as insecticides worldwide or as warfare agents. Thousands of cases of acute poisoning have been reported5. This acute toxicity inhibits AChE at nerve terminals where inhibition causes accumulation of ACh. This, in turn, induces over- stimulation of nicotinic and muscarinic receptors in the central and peripheral nervous systems and the consequent signs and symptoms6