Azucares Reductores

ab102523 Amylase Assay Kit

Instructions for Use
For the rapid, sensitive and accurate measurement of Amylase activity in various samples. This product is for research use only and is not intended for diagnostic use.

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Table of Contents
1. 2. 3. 4. 5. 6. Overview Protocol Summary Components and Storage Assay Protocol Data Analysis Troubleshooting 3 4 5 7 9 11

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1. OverviewAmylases are enzymes that break starch down to sugar molecules. α-amylase is the major form of amylase found in humans and other mammals as well as an enzyme present in seeds, or in fungi (baker’s yeast for instance). α-amylase is a calcium metalloenzyme, completely unable to function in the absence of calcium. In Human physiology, both the salivary and pancreatic amylases are major digestiveenzymes. Increased enzyme levels in Humans are associated with salivary trauma; mumps due to inflammation of the salivary glands, pancreatitis and renal failure. A simple, direct and automation-ready procedure for measuring α-amylase activity is, therefore, very desirable. Abcam’s α-amylase Assay uses ethylidene-pNP-G7 as the

substrate. Once the substrate has been specifically cleaved by αamylase, thesmaller fragments produced can be acted upon by αglucosidase, which causes the ultimate release of the chromophore that can then be measured at 405 nm. The assay can detect αamylase content as low as 0.2 mU.

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2. Protocol Summary
Sample Preparation

Standard Curve Preparation A Add Reaction Mix

Measure Optical Density

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3. Components and Storage
A. Kit Components
ItemQuantity

Amylase Assay Buffer Amylase Substrate Mix Amylase Positive Control (Lyophilized) Nitrophenol standard (2mM)

25 mL 5 mL 1 vial 150 µL

* Store kit at -20° C. • • • • Warm the assay buffer to room temperature before use. Briefly centrifuge vials before opening. Read the entire protocol before performing the assay. Keep samples and Amylase Positive Control on ice during the assay.

5B. Additional Materials Required • • • • • Microcentrifuge Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96 well plate Orbital shaker

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4. Assay Protocol
1. Sample Preparation: Dissolve Amylase Positive Control into 50 µl Assay Buffer, and store at -20°C. a. For serum or urine samples: Serum and urine samples can be tested directly: Add 0.5-50 µl samples or 5 µlAmylase Positive Control into each well, and adjust volume to 50 µl with dH2O. b. For tissue or cell samples: Tissue (100 mg) or cells (4 x 10 ) can be extracted with 0.5 ml Assay Buffer and centrifuged at 16,000 x g for 10 min. The clear extract can be assayed directly. For unknown samples, we suggest using different doses to ensure the readings are within the linear range. 2. Standard CurvePreparation: Add 0, 2, 4, 6, 8, 10 µl of 2 mM nitrophenol Standard mix into 96-well plate in duplicate to generate 0, 4, 8, 12, 16, 20 nmol/well nitrophenol standard. Bring the total volume to 50 µl with dH2O. 3. Reaction Mix: Prepare enough reaction mix for samples, standard and positive control. For each reaction: Assay Buffer Substrate Mix 50 µl 50 µl
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4. Add 100 µl of the reaction mixinto each reaction and mix. Measure immediately (T0) at OD405nm to get ODT0. Incubate the reaction at 25° for various times (T1) and measure OD405nm to get C ODT1 Note: Sample incubation time can vary depending on α-amylase activity in samples. We recommend observing the reaction kinetics then choosing the linear range for T0-T1. The Standard Curve will not change as incubation time increases.8

5. Data Analysis
Plot the Nitrophenol standard curve. Apply the ∆OD (∆OD = ODT1 – ODT0) to the Nitrophenol standard curve to get B nmol of Nitrophenol generated by amylase between T0 and T1.
B x Sample Dilution Factor Amylase Activity = ∆T x V = nmol/min/ml = mU/ml

Where: B is the Nitrophenol amount from Standard Curve (in nmol). ∆T is the time between T0 and T1 (in min). V is the...