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alpha-Amylase Assay Kit

AcA Blood, Grains, Saliva, Urine
Catalog No. ABIN1000295
  • Target See all Amylase, alpha (AMY) Kits
    Amylase, alpha (AMY)
    Application
    Activity Assay (AcA)
    Sample Type
    Blood, Saliva, Urine, Grains
    Specificity
    0.3 U/L
    Characteristics
    Sensitive and accurate. Linear detection range 0.3 to 50 U/L - amylase in 96-well plate assay.
    Convenient. The procedure involves adding a single working reagent, incubation for 15 min, followed by the detection reagent and a 20-min incubation and reading the optical density at 585 nm.
    Components
    Assay Buffer (pH 7.0): 20 mL. Substrate: 120 µL. Detection Reagent: 20 mL. Enzyme A: 120 µL. Glucose Standard: 1 mL. Enzyme B: 120 µL.
    Material not included
    Pipeting devices, centrifuge tubes, clear flat-bottom 96-well plates, plate reader, and optionally membrane filters (e.g. Microcon YM-10 from Millipore).
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  • Application Notes
    Determination of -amylase activity in blood, saliva, urine, grains and other agricultural samples.
    Comment

    It is prudent to perform a pilot test with samples at various dilutions. Recommended dilution: serum 50-fold, saliva 2,000-fold in Assay Buffer prior to assay.
    1. Prepare 400 μM Glucose Standard by mixing 10 μL of the provided (300 mg/dL) standard with 406 μL Assay Buffer. Transfer 10 μL Assay Buffer, 10 μL 400 μM glucose, and 10 μL of each sample into separate wells of a clear flat-bottom 96-well plate.
    2. Prepare enough Working Reagent for each well by mixing 40 μL Assay Buffer, 0.5 μL Substrate, 1 μL Enzyme A, 1 μL Enzyme B. Transfer 40 μL Working Reagent to each well. Incubate for 15 min at room temperature (25 °C).
    3. Add 150 μL Detection Reagent to each well. Mix and incubate for 20 min at room temperature (25 °C). Read OD585nm (540-610nm) on a plate reader.

    Reagent Preparation

    Equilibrate all components to room temperature. Keep thawed Enzyme Mix in a refrigerator or on ice. The substrate may have precipitates. Prior to use, vortex tube to dissolve precipitates, gentle swirl the Detection Reagent bottle.

    Sample Preparation

    Ideally samples are assayed fresh. When stored frozen, -amylase is stable for one month. Ascorbic acid, heparin, EDTA, EGTA, citrate, SDS, Tris (> 8mM) and ethanol (>0.4%) interfere and should be avoided in sample preparation.

    Restrictions
    For Research Use only
  • Storage
    -20 °C
  • Target
    Amylase, alpha (AMY)
    Alternative Name
    alpha-Amylase (AMY Products)
    Synonyms
    LOC663954 Kit, LOC100136478 Kit, alpha amylase Kit, alpha-amylase Kit, LOC663954 Kit, HQ_RS05305 Kit, LOC100136478 Kit
    Background
    Quantitative determination of alpha-amylase activity based on colorimetric glucose determination at 585nm.
    Procedure: 40 min.

    Amylase belongs to the family of glycoside hydrolase enzymes that break down starch into glucose molecules by acting on alpha-1,4- glycosidic bonds. The alpha-amylases (EC 3.2.1.1) cleave at random locations on the starch chain, ultimately yielding maltotriose and maltose, glucose and limit dextrin from amylose and amylopectin. In mammals, alpha-amylase is a major digestive enzyme. Increased enzyme levels in humans are associated with salivary trauma, mumps due to inflammation of the salivary glands, pancreatitis and renal failure. Simple, direct and automation-ready procedures for measuring amylase activity are very desirable. This alpha-amylase assay method involves two steps: (1). alpha-amylase in the sample hydrolyzes starch and the product is rapidly converted to glucose by alpha-glucosidase and hydrogen peroxide by glucose oxidase, (2). hydrogen peroxide concentration is determined with a colorimetric reagent.
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