alpha-Amylase Assay Kit

Catalog No. ABIN1000295

Quick Overview for alpha-Amylase Assay Kit (ABIN1000295)

Target: Amylase, alpha (AMY)

Application: Activity Assay (AcA)

Sample Type: Blood, Saliva, Urine, Grains

Product Details

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).

Application Details

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

Handling

Storage: -20 °C

Target Details

Target: Amylase, alpha (AMY)

Alternative Name: alpha-Amylase

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.