Enzymatic Method for Determining Amylase Activity (Amylase Activity Assay)

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.

Precautions and Disclaimer

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.

Reagents

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

Reagents and Equipment Required but Not Provided:

96 well flat-bottom plate – It is recommended to use clear plates (Catalog Number M4436 or equivalent) for colorimetric assays.

Spectrophotometric multiwell plate reader

Preparation Instructions

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.

Storage/Stability

The kit is shipped on wet ice. Storage at –20 °C, protected from light, is recommended.

Procedure

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.

Sample Preparation
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

Assay Reaction

1. Prepare the Master Reaction Mix according to Table 1. 100 µL of the Master Reaction Mix is required for each reaction (well).

2. Add 100 µL of the Master Reaction Mix to each of the sample, standard, and positive control wells. Mix well using a horizontal shaker or by pipetting.
   
   
3. After 2–3 minutes (T_initial), measure the absorbance at 405 nm (A₄₀₅)_initial
   Note: It is essential that (A₄₀₅)_initial is in the linear range of the standard curve.
   
   
4. Incubate the plate at 25 °C, measuring the absorbance (A₄₀₅) every 5 minutes. Protect the plate from light during the incubation.
   
   
5. Continue taking measurements until the value of the most active sample is greater than the value of the highest standard (20 nmole/well). At this time, the most active sample is near or exceeds the end of the linear range of the standard curve.
   
   
6. The final absorbance measurement [(A₄₀₅)_final] for calculating the enzyme activity would be the penultimate reading or the value before the most active sample is near or exceeds the end of the linear range of the standard curve (see step 5). The time of the penultimate reading is T_final
   Note: It is essential the final measurement falls within the linear range of the standard curve.

Results

Calculations

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 T_initial to T_final 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 T_initial to T_final

The amylase activity of a sample may be determined by the following equation:

B = Amount (nmole) of nitrophenol generated between T_initial and T_final

Reaction Time = T_final – T_initial (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.