Alcohol dehydrogenase (ADH) ELISA Kit

Antigen: Alcohol Dehydrogenase (ADH)

Synonyme: ALCOHOL DEHYDROGENASE, ARABIDOPSIS TAPETUM 1, ASD, ATA1, ADH, ADH1, ALCOHOL DEHYDROGENASE 1, ARABIDOPSIS THALIANA ALCOHOL DEHYDROGENASE, ATADH, ATADH1, F22K20.19, F22K20_19, BG:DS01486.8, CG32954, DmADH, Dreg-1, Reg-1, T16, dADH, DmelCG3481, CG3481, Adh-2, dvir_GLEANR_2772, DvirGJ18209, GJ18209, Adh2, Adh-1, DvirAdh, dvir_GLEANR_2771, DvirGJ18208, GJ18208, Adh1

Reaktivität: Ratte (Rattus)

Applikation: ELISA

Zertifikate: ISO 9001:2008

Produktnummer: ABIN367549

Produktbeschreibung

Produktmerkmale: This immunoassay kit allows for the in vitro quantitative determination of rat ADH concentrations in serum, plasma and other biological fluids.

Beschreibung: Alcohol dehydrogenase (ADH) is an enzyme discovered in the mid-1960s in Drosophila melanogaster. Since then, there has been extensive research on the enzyme. Alcohol dehydrogenase is a dimer, weighing 80 kDa. Alcohol dehydrogenases are a group of seven dehydrogenase enzymes that occur in many organisms and facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD + to NADH. In humans and many other animals, they serve to break down alcohols which could otherwise be toxic, in yeast and many bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation. In humans, alcohol is metabolized by a rate of 1 ounce or 7 to 10 g/hour. Alcohol dehydrogenase is responsible for catalyzing oxidation of primary and secondary alcohols to aldehydes and ketones, respectively, and also can effect the reverse reaction. It does not work well with primary alcohols. Instead, it works the best with secondary and cyclic alcohols. In humans, it exists in multiple forms as a 3 dimer and is encoded by at least seven different genes. There are five classes (I-V) of alcohol dehydrogenase, but the hepatic form that is primarily used in humans is class 1. Class 1 consists of A,B, and C subunits that are encoded by the genes ADH1A, ADH1B, and ADH1C. The enzyme is contained in the lining of the stomach and in the liver. It catalyzes the oxidation of ethanol to acetaldehyde. This allows the consumption of alcoholic beverages, but its evolutionary purpose is probably the breakdown of alcohols naturally contained in foods or produced by bacteria in the digestive tract. Alcohol dehydrogenase is also involved in the toxicity of other types of alcohol: for instance, it oxidizes methanol to produce formaldehyde and ethylene glycol to ultimately yield glycolic and oxalic acids. Humans have at least six slightly different alcohol dehydrogenases. All of them are dimers (consist of two polypeptides), with each dimer containing two zinc ions Zn 2+ . One of those ions is crucial for the operation of the enzyme: it is located at the catalytic site and holds the hydroxyl group of the alcohol in place. 4

Spezifität: This assay recognizes recombinant and natural rat ADH. No significant cross-reactivity or interference was observed.

Synonyme: ALCOHOL DEHYDROGENASE, ARABIDOPSIS TAPETUM 1, ASD, ATA1, ADH, ADH1, ALCOHOL DEHYDROGENASE 1, ARABIDOPSIS THALIANA ALCOHOL DEHYDROGENASE, ATADH, ATADH1, F22K20.19, F22K20_19, BG:DS01486.8, CG32954, DmADH, Dreg-1, Reg-1, T16, dADH, DmelCG3481, CG3481, Adh-2, dvir_GLEANR_2772, DvirGJ18209, GJ18209, Adh2, Adh-1, DvirAdh, dvir_GLEANR_2771, DvirGJ18208, GJ18208, Adh1

Anwendungen

Prinzip: The microtiter plate provided in this kit has been pre-coated with an antibody specific to ADH. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for ADH and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain ADH, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm±2 nm. The concentration of ADH in the samples is then determined by comparing the O.D. of the samples to the standard curve. 5

Protokoll: Preparation of Reagents: Bring all reagents to room temperature before use. 1. Wash Buffer If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. 2. Standard Reconstitute the Standard with 1.0 ml of Sample Diluent. This reconstitution produces a stock solution of 25 ng/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (25 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml). 3. Biotin-antibody Dilute to the working concentration using Biotin-antibody Diluent(1:100), respectively. 8 4. HRP-avidin Dilute to the working concentration using HRP-avidin Diluent(1:100), respectively. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. Sample Collection and Storage: Cell Culture Supernates Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. 9 Remove serum and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed samples are not suitable for use in this assay. Assay Procedure: Calculation of Results: Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the ADH concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Limitations of the Procedure: 12 The kit should not be used beyond the expiration date on the kit label. Do not mix or substitute reagents with those from other lots or sources. It is important that the Calibrator Diluent selected for the standard curve be consistent with the samples being assayed. If samples generate values higher than the highest standard, dilute the samples with the appropriate Calibrator Diluent and repeat the assay. Any variation in Standard Diluent, operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding. This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Applikationshinweise: 13 When mixing or reconstituting protein solutions, always avoid foaming. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, and/or rotating the plate 180 degrees between wash steps may improve assay precision. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue. Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution. 14

Bestandteile: Reagent Quantity Assay plate 1 Standard 2 Sample Diluent 1 x 20 ml Biotin-antibody Diluent 1 x 10 ml HRP-avidin Diluent 1 x 10 ml Biotin-antibody 1 x 120µl HRP-avidin 1 x 120µl Wash Buffer 1 x 20 ml (25×concentrate) TMB Substrate 1 x 10 ml Stop Solution 1 x 10 ml STORAGE 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 7 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Lagerung: 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 7 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement. Expiration date: six months from the date of manufacture.

Forschungsgebiet: Enzyme

Beschränkungen: Nur für Forschungszwecke einsetzbar