This assay protocol is suitable for the colorimetric detection of Amylase activity in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples using the Amylase Activity Assay Kit (MAK009). Amylase activity is determined using a coupled enzymatic assay, which results in a colorimetric (405 nm) product, proportional to the amount of substrate, ethylidene-pNP-G7, cleaved by the amylase. One unit is the amount of amylase that cleaves ethylidene-pNP-G7 to generate 1.0 µmole of p-nitrophenol per minute at 25 °C.
This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.
Amylase Assay Buffer 25 mL
Catalog Number MAK009A
Amylase Substrate Mix 5 mL
Catalog Number MAK009B
Amylase Positive Control 1 vL
Catalog Number MAK009C
Nitrophenol Standard 150 µL
Catalog Number MAK009D
96 well flat-bottom plate – It is recommended to use clear plates (Catalog Number M4436 or equivalent) for colorimetric assays.
Spectrophotometric multiwell plate reader
Briefly centrifuge vials before opening. Use ultrapure water for the preparation of reagents. To maintain reagent integrity, avoid repeated freeze/thaw cycles.
Amylase Assay Buffer – Allow buffer to come to room temperature before use.
Amylase Positive Control – Reconstitute with 50 µL of Amylase Assay Buffer. Mix well by pipetting, then aliquot and store at –20 °C. Use within 2 months of reconstitution.
The kit is shipped on wet ice. Storage at –20 °C, protected from light, is recommended.
All samples and standards should be run in duplicate.
Nitrophenol Standards for Colorimetric Detection
Add 0, 2, 4, 6, 8, 10 µL of the 2 mM Nitrophenol Standard into a 96 well plate, generating 0 (blank), 4, 8, 12, 16, and 20 nmole/well standards. Add water to each well to bring the volume to 50 µL.
Tissue (100 mg) or cells (4 x106) can be homogenized in 0.5 mL of the Amylase Assay Buffer. Centrifuge the samples at 13,000 x g for 10 minutes to remove insoluble material.
Serum and urine samples can be directly added to wells.
Add 1–50 µL of sample into wells of a 96 well plate. Bring samples to a final volume of 50 µL with Amylase Assay Buffer.
Note: For unknown samples, it is suggested to test several sample dilutions to ensure the readings are within the linear range of the standard curve.
For the positive control (optional), add 5 µL of the Amylase Positive Control solution to wells and adjust to 50 µL with the Amylase Assay Buffer
Correct for the background by subtracting the final measurement (A405)final obtained for the 0 (blank) nitrophenol standard from the (A405)final measurement of the standards and samples.
Note: A new standard curve must be set up each time the assay is run.
Calculate the change in absorbance from Tinitial to Tfinal for the samples.
∆A405 = (A405)final – (A405)initial
Compare the ∆A405 of each sample to the standard curve to determine the amount of nitrophenol (B) generated by the amylase between Tinitial to Tfinal
The amylase activity of a sample may be determined by the following equation:
|Amylase Activity =|| B x Sample Dilution Factor
(Reaction Time) x V
B = Amount (nmole) of nitrophenol generated between Tinitial and Tfinal
Reaction Time = Tfinal – Tinitial (minutes)
V = sample volume (mL) added to well
Amylase activity reported as nmole/min/mL. One unit of amylase is the amount of amylase that cleaves ethylidene-pNP-G7 to generate 1.0 µmole of
p-nitrophenol per minute at 25 °C.
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