α-Amylase, an endohydrolase, hydrolyzes α-1, 4-glucosidic bonds in α-glucans and starch into glucose while yielding maltotriose and maltose. Fungal and bacterial α-Amylase derivatives have ultimately been a primary source in industrial and food industries. Mammalian α-Amylase is critical in digestion, glucose-regulation in blood, and is an important indicator in the pancreas for pancreatitis and pancreatic cancer. Monitoring its levels is important in understanding overall health for underlying conditions like kidney disease, diabetes, and cystic fibrosis.

α-Amylase Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring α-Amylase activity in plasma, serum, cell lysates, urine, saliva, and other biological liquid samples. This assay is based on spectrophotometric detection of the chromophore 2-chloro-4-nitrophenol after cleavage from the labeled substrate. The light emitted from 2-chloro-4-nitrophenol can be quantified using a spectrophotometer or microplate reader at 405 nm. The change in absorbance of the 2-chloro-4-nitrophenol is directly proportional to α-Amylase activity in the sample.

• Microplate reader capable of measuring absorbance at 405 nm at 25oC
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• 5 mL tubes
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 50 µL of the samples and positive control to the 96-well microtiter plate. Include substrate background wells by adding Assay Buffer only.
2. Add 100 µL of the diluted Nitrophenol standards to 96-well microtiter plate.
3. Initiate the reaction by adding 50 µL diluted α-Amylase substrate solution to each well. Carefully shake the plate for a few seconds to mix well.
4. For kinetic reading, immediately start measuring absorbance at 405 nm continuously at 25°C and record data every 4 minutes for 32 minutes.