a-Amylase Assay Kit, BioAssay™, High Sensitivity

Amylase belongs to the family of glycoside hydrolase enzymes that break down starch into glucose molecules by acting on a-1,4- glycosidic bonds. The a-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, a-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. a-Amylase assay method involves two steps: (1). a-Amylase in the sample hydrolyzes starch and the product is rapidly converted to glucose by a-glucosidase and hydrogen peroxide by glucose oxidase; (2). hydrogen peroxide concentration is determined with a colorimetric reagent.||Applications:|Determination of a-amylase activity in blood, saliva, urine, grains and other agricultural samples.||Key Features:|Sensitive and accurate. Linear detection range 0.3 to 50 U/L a- 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.||Kit Contents:|Assay Buffer ,pH 7.0, 1x20ml |Detection Reagent, 1x20ml |Glucose Standard, 1x1ml |Substrate, 1x120ul|Enzyme A, 1x120ul|Enzyme B, 1x120ul||Storage and Stability:|Store Detection Reagent at 4°C. Store other components at 4°C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.  ||Materials Required, But Not Provided:|Pipeting devices |centrifuge tubes |clear flat-bottom 96-well plates |plate reader |membrane filters (optional)||Procedure:|Reagents: 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; gentlly swirl the Detection Reagent bottle. ||Sample preparation: Ideally samples are assayed fresh. When stored frozen, a-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. If glucose is present in the sample, treat the samples as described in General Considerations. 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 400uM Glucose Standard by mixing 10ul of the provided (300 mg/dL) standard with 406ul Assay Buffer. Transfer 10ul Assay Buffer, 10ul 400uM glucose, and 10ul of each sample into separate wells of a clear flat-bottom 96-well plate.|2. Prepare enough Working Reagent for each well by mixing 40ul Assay Buffer, 0.5ul Substrate, 1ul Enzyme A, 1ul Enzyme B. Transfer 40ul Working Reagent to each well. Incubate for 15 min at room temperature (25°C).|3. Add 150ul Detection Reagent to each well. Mix and incubate for 20 min at room temperature (25°C). |4. Read OD585nm (540-610nm) on a plate reader.||Calculation:|The Amylase activity is calculated as||Activity = OD(sample) - OD(buffer) x 400 x n (U/L) | OD(standard) -OD(buffer) t(min)||OD(sample), OD(standard) and OD(buffer) are optical density values of the sample, the 400uM glucose standard and Assay Buffer. t is the incubation time. t=15 min in the standard protocol. n is the dilution factor (n=50 for serum, 2000 for saliva). One unit of enzyme catalyzes the production of 1uMole of glucose per min under the assay conditions.|Note: if the calculated activity is higher than 50 U/L, dilute sample in Assay Buffer and repeat assay. Multiply the results by the dilution factor.||General Considerations:|For samples known to contain glucose, use a membrane filter to remove glucose: |1. Load 50ul sample in a 10 kD cutoff membrane filter and add 500ul Assay Buffer. |2. Centrifuge at 14000 rpm for 30 min, check level of sample, ideally the sample level will be less than 50ul. |3. Add 500ul Assay Buffer and repeat the centrifugation. Measure final sample volume with a pipetman and calculate dilution factor n=final sample volume/50ul.