Description
Arginase Inhibitor Screening Kit (BA0193) (BA0193)
The Arginase Inhibitor Screening Kit (SKU: BA0193) provides a sensitive and convenient means of screening for inhibitors of arginase, the enzyme that catalyses the conversion of arginine to ornithine and urea. Arginase is expressed predominantly in the liver, with lesser expression in breast, kidney, testes, salivary glands, epidermis and erythrocytes, and its activity is important for protection against ammonia toxicity as well as for cell growth and repair. Because excessive arginase activity has been linked to cardiovascular and neural disorders, arginase inhibitors are of considerable interest in research and drug discovery. This kit utilises a chromogen that forms a coloured complex specifically with the urea generated in the arginase reaction, giving a signal directly proportional to enzyme activity. The percent inhibition of a test compound is calculated by comparing a pre-incubated reaction against an untreated control. The homogeneous, non-radioactive procedure can be completed in under two hours without the need for a 37°C heater.
| Product Name: | Arginase Inhibitor Screening Kit (BA0193) |
| SKU: | BA0193 |
| Detection Method: | Colorimetric detection at 430 nm. The kit employs a chromogen that forms a coloured complex specifically with the urea produced in the arginase reaction; colour intensity is directly proportional to arginase activity. Percent inhibition is determined by comparing a reaction pre-incubated with a test compound against an untreated control reaction. |
| Sample Type: | Purified arginase reactions; test compounds dissolved in the solvent of choice (e.g. DMSO) |
| Species Reactivity: | All |
| Assay Time: | Less than 2 hours |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store the Arginine Buffer at -20°C and all other components at 2-8°C. |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
This assay measures arginase inhibition using a chromogen that reacts specifically with the urea produced when arginase converts arginine to ornithine and urea. The intensity of the resulting colour, read at 430 nm, is directly proportional to arginase activity. By comparing the signal of a reaction pre-incubated with a test compound against an untreated control, the percent inhibition of the compound can be determined. Neither the arginase enzyme nor a control inhibitor is supplied with the kit.
- Safe. Non-radioactive assay.
- High-throughput. Homogeneous mix-incubate-measure type assay that can be readily automated on an HTS liquid-handling system.
- Rapid and reliable. Can be completed in less than 2 hours with no 37°C heater required.
- High-throughput screening for inhibitors and evaluation of arginase inhibitors.
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 | Bring all reagents to room temperature prior to assay. Prepare the Substrate Buffer and Urea Reagent freshly and use within 2 hours. |
| 2 | Sample preparation: dissolve the test compounds in the solvent of choice. First test the tolerance of the solvent for the enzyme. For human arginase I, dilute purified arginase to 0.0012 U/µL using dH2O. For other species, experimentally determine the Km and adjust the Arginine Buffer volume so the final substrate concentration in the 50 µL reaction is near the Km. |
| 3 | Transfer 40 µL of arginase into separate wells. |
| 4 | Reserve two wells with arginase for the Blank and Control. |
| 5 | To the Control and Blank wells, add 5 µL of the solvent in which the test compounds are dissolved (e.g. 5 µL 100 v/v% DMSO). |
| 6 | To the remaining arginase wells, add 5 µL of the test compounds. Tap plate and mix. |
| 7 | Incubate the plate for 15 minutes at 25°C. |
| 8 | Prepare sufficient Substrate Buffer by combining, per well, 4 µL Arginine Buffer and 2 µL Mn Solution. Add 5 µL Substrate Buffer to all sample wells except the Blank; add 5 µL dH2O to the Blank. Tap and mix, then incubate 30 minutes at 25°C. |
| 9 | Urea determination: prepare Urea Reagent by combining equal volumes of Reagent A and Reagent B. Add 200 µL Urea Reagent to all wells (this stops the arginase reaction). Tap to mix, incubate 60 minutes at room temperature and read optical density at 430 nm. |
% Inhibition = (1 - (dODTestCpd / dODNoInhibitor)) x 100%, where dODTestCpd is the OD430nm of a test compound minus the OD430nm of the Blank well (no substrate) at 60 min, and dODNoInhibitor is the OD430nm of the Control well (no inhibitor) minus the OD430nm of the Blank well (no substrate) at 60 min.
| Component | Quantity | Storage |
| Arginine Buffer (pH 9.5) | 1 mL | -20°C |
| Mn Solution | 300 µL | 2-8°C |
| Reagent A | 12 mL | 2-8°C |
| Reagent B | 12 mL | 2-8°C |
