Description
Alpha-Amylase Assay Kit (BA0082) (BA0082)
The Alpha-Amylase Assay Kit (SKU: BA0082) provides a simple, direct and automation-ready procedure for measuring alpha-amylase activity. Amylase belongs to the glycoside hydrolase family and breaks down starch into glucose by acting on alpha-1,4-glycosidic bonds; in mammals it is a major digestive enzyme, and elevated levels are associated with salivary trauma, mumps, pancreatitis and renal failure. This assay involves two steps: alpha-amylase hydrolyses starch and the product is rapidly converted to glucose and hydrogen peroxide, then the hydrogen peroxide concentration is determined with a colorimetric reagent. The optical density at 585 nm is proportional to the alpha-amylase activity in the sample.
| Product Name: | Alpha-Amylase Assay Kit (BA0082) |
| SKU: | BA0082 |
| Detection Method: | Colorimetric |
| Detection Range: | 0.3 to 50 U/L |
| Sample Type: | Blood, saliva, urine, grains and other agricultural samples |
| Species Reactivity: | All |
| Assay Time: | 15 minute incubation followed by 20 minute detection |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
Quantitative colorimetric determination of alpha-amylase activity at 585 nm. Amylase hydrolyses starch to glucose, which is oxidised to hydrogen peroxide and quantified with a colorimetric reagent.
- Sensitive and accurate, with a linear detection range of 0.3 to 50 U/L in a 96-well plate assay
- Convenient single-working-reagent procedure
- Optical density read at 585 nm
- Suitable for a range of biological and agricultural samples
- Determination of alpha-amylase activity in blood, saliva, urine, grains and other agricultural samples
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Reagents. Equilibrate all components to room temperature and keep thawed enzyme in a refrigerator or on ice. Vortex the substrate to dissolve any precipitates and gently swirl the Detection Reagent bottle. |
| 2 | Sample preparation. Assay samples fresh where possible; when stored frozen, alpha-amylase is stable for one month. Avoid ascorbic acid, heparin, EDTA, EGTA, citrate, SDS, Tris above 8 mM and ethanol above 0.4%. Recommended dilutions are serum 50-fold and saliva 2,000-fold in Assay Buffer; perform a pilot test at various dilutions. |
| 3 | Prepare 400 uM glucose standard by mixing 10 uL of the provided 300 mg/dL standard with 406 uL Assay Buffer. Transfer 10 uL Assay Buffer, 10 uL 400 uM glucose and 10 uL of each sample into separate wells of a clear flat-bottom 96-well plate. |
| 4 | Prepare enough working reagent by mixing, per well, 40 uL Assay Buffer, 0.5 uL Substrate, 1 uL Enzyme A and 1 uL Enzyme B. Transfer 40 uL working reagent to each well and incubate for 15 minutes at room temperature (25C). |
| 5 | Add 150 uL Detection Reagent to each well, mix and incubate for 20 minutes at room temperature (25C). Read OD585nm (540-610 nm) on a plate reader. |
Activity (U/L) = [(OD-sample - OD-buffer) / (OD-STD - OD-buffer)] x (400 / t) x n, where OD-sample, OD-STD and OD-buffer are the optical density values of the sample, the 400 uM glucose standard and Assay Buffer; t is the incubation time (15 min in the standard protocol); and n is the dilution factor (50 for serum, 2000 for saliva). One unit of enzyme catalyses the production of 1 umole of glucose per minute. If the calculated activity is higher than 50 U/L, dilute the sample in Assay Buffer, repeat and multiply by the dilution factor.
| Component | Quantity | Storage |
| Assay Buffer (pH 7.0) | 20 mL | -20C |
| Detection Reagent | 20 mL | -20C |
| Glucose Standard | 1 mL | -20C |
| Substrate | 120 uL | -20C |
| Enzyme A | 120 uL | -20C |
| Enzyme B | 120 uL | -20C |
