| Micro Assay for Flour Alpha Amylase Activity |
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Micro Assay for Flour Alpha Amylase Activity
Adapted by Mary Guttieri for the Soft Wheat Quality Laboratory
The new method adapts the AACC Method 22-02 using the Ceralpha K-CERA (Megazyme) alpha-amylase assay procedure for higher throughput to determine flour alpha-amylase activity in a microwell plate. All reagents, controls and precautions are as described in the Megazyme manual.
Required Materials
- Ceralpha Alpha-Amylase Kit (AACC Method 22-02)
- 50 mL conical centrifuge tubes
- Centrifuge with rotor to spin 50 mL conical tubes at 1000 xg
- Analytical balance
- Microplate reader and plate (510 nm)
- Vortex mixer
- Water bath at 40o C
- Multichannel repeating pipette
Ceralpha Substrate and Stopping Reagent
Ceralpha substrate is prepared as described and stored frozen (-20oC) in 1 mL aliquots in microcentrifuge tubes.
Additional Stopping Reagent is prepared using 1% w/v sodium phosphate tribasic dodecahydrate in distilled water adjusted to pH11.
Enzyme Extraction
- Accurately weigh 3.0 g of ground grain or flour into a 50 mL conical centrifuge tube.
- Add 20.0 mL of 1X Extraction Buffer solution (pH 5.4) to each tube and mix vigorously.
- Allow enzyme to extract over 20 minutes in a 40?C water bath, with occasional mixing.
- Centrifuge 1,000 x g for ten minutes.
- Assay enzyme activity within two hours.
Reaction Blank
A single set of triplicate Reaction Blanks (non-enzymatic control) is prepared as follows for each batch of samples being analyzed.
- 0.3 mL of stopping reagent
- 20 ?L of substrate solution at the start of the reaction time
- 20 ?L of any enzyme preparation in the sample set
The mean absorbance of the non-enzymatic control is subtracted from all assays conducted during that day to establish the background or blank absorbance.
Assay Procedure
- Dispense 20 ?L aliquots of Ceralpha Reagent Solution into a microtiter plate and pre-incubate the tubes and contents at 40?C for 5 min. Dispense 3 aliquots for each enzyme extract (assay each extract in triplicate).
- To each well containing Ceralpha Reagent solution (20 ?L), add 20 ?L of wheat ?-amylase extract directly to the bottom of the well at 30 second intervals.
- Incubate at 40?C for exactly twenty min from time of addition.
- Following the 20 min incubation period, add exactly 0.3 mL of Stopping Reagent.
- Read the absorbance of the solutions and the reaction blank at 400 nm against 340 ?L distilled water.
