Description
alpha-Amylase Inhibitor Screening Kit (BA0257) (BA0257)
The alpha-Amylase Inhibitor Screening Kit (SKU: BA0257) provides a robust, high-throughput fluorescence polarisation method for identifying inhibitors of alpha-amylase. Amylases belong to the glycoside hydrolase family and break down starch into glucose by acting on alpha-1,4-glycosidic bonds; the alpha-amylases cleave at random locations on the starch chain to yield maltotriose, maltose, glucose and limit dextrin. In mammals alpha-amylase is a major digestive enzyme, and increased levels in humans are associated with salivary trauma, mumps, pancreatitis and renal failure. This kit uses fluorescence polarisation, a highly reliable technique that significantly reduces background matrix interferences, in which alpha-amylase cleaves a fluorescent amylose substrate to decrease FP; inhibition is therefore reflected by an increase in FP. The homogeneous mix-incubate-measure assay can be completed in under an hour at room temperature, exhibits a Z-prime factor above 0.90 in 384-well format and can be readily automated for thousands of samples per day.
| Product Name: | alpha-Amylase Inhibitor Screening Kit (BA0257) |
| SKU: | BA0257 |
| Detection Method: | Fluorescence polarisation (FP) at lambda ex/em = 485/520 nm. alpha-Amylase cleaves a fluorescent amylose substrate, decreasing FP; inhibition is determined by the increase in FP, which is proportional to reduced enzyme activity. |
| Sample Type: | Purified alpha-amylase reactions; test compounds dissolved in H2O or a solvent of choice (e.g. DMSO) |
| Species Reactivity: | All |
| Assay Time: | Under 1 hour |
| Kit Size: | 400 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all components at -20°C upon receipt. |
| Shelf Life: | 6 months after receipt |
| Shipping: | Room Temperature |
This assay screens for alpha-amylase inhibitors using fluorescence polarisation. alpha-Amylase cleaves a fluorescent amylose substrate, decreasing the fluorescence polarisation signal (lambda ex/em = 485/520 nm); in the presence of an inhibitor, less substrate is cleaved and the FP remains higher. The change in FP relative to control and blank wells is used to calculate the percentage of enzyme activity remaining, and hence inhibition, for each test compound.
- Safe. Non-radioactive assay.
- Fast and convenient. Homogeneous mix-incubate-measure assay that can be completed in under an hour at room temperature.
- Robust and high-throughput. The FP assay greatly reduces background matrix interferences; a Z-prime factor above 0.90 was observed in a 384-well format, and it can be readily automated for thousands of samples per day.
- Evaluation of drugs and screening of potential inhibitors of alpha-amylase.
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 | Equilibrate all components to room temperature and keep the enzyme on ice; briefly centrifuge the Amylase and inhibitor control. Assays are performed in black flat-bottom plates with a reader capable of measuring FP at 485/520 nm. The 384-well procedure below can be scaled for 96-well assays (30 µL enzyme, 10 µL inhibitor, 60 µL Substrate). |
| 2 | Dissolve test compounds in H2O or a solvent of choice (e.g. DMSO), first testing the tolerance of the solvent for the enzyme. |
| 3 | Pre-incubation: dilute the provided purified human salivary alpha-amylase 10,000-fold in Assay Buffer (or 20 mM potassium phosphate, 50 mM NaCl, pH 7.0) and use within 30 minutes. Transfer 15 µL of diluted enzyme into Test wells, and include a Control well with 15 µL enzyme and a Blank well with 15 µL Assay Buffer. |
| 4 | Add 5 µL of H2O or the solvent used for the test compounds to the Control and Blank wells, and 5 µL of the test compounds to the Test wells. Tap the plate to mix and incubate for 10 minutes at room temperature to allow the inhibitor to block enzyme activity. |
| 5 | Enzyme activity assay: add 30 µL of the Substrate to all wells and tap briefly to mix. Incubate for 30 minutes at room temperature protected from light, then read the FP at lambda ex/em = 485/520 nm. |
Enzyme Activity (%) = ((FP_Blank - FP_Compound) / (FP_Blank - FP_Control)) x 100%, where FP_Compound, FP_Control and FP_Blank are the fluorescence polarisation values of the test compound, Control and Blank wells respectively. Inhibition is derived from the reduction in enzyme activity.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20°C |
| Amylase | 30 µL | -20°C |
| Substrate | 12 mL | -20°C |
| Inhibitor Control (3 mM Acarbose) | 80 µL | -20°C |
