Description
Arginase Activity Assay Kit (Colorimetric) (BA0016) (BA0016)
The Arginase Activity Assay Kit (Colorimetric) (SKU: BA0016) provides a sensitive, convenient colorimetric method for determining arginase activity in biological samples. Arginase catalyses the conversion of arginine to ornithine and urea, completing the final step of the urea cycle, and raised blood arginase activity is associated with liver damage while arginase deficiency causes hyperargininemia. The method uses a chromogen that forms a coloured complex specifically with the urea produced in the arginase reaction, so the colour intensity is directly proportional to arginase activity. With a detection limit of 0.3 U/L for a 2-hour reaction in the 96-well format, the assay is well suited to enzyme preparations, serum, plasma and tissue culture samples. Its simple microplate procedure can be readily automated for high-throughput measurement of thousands of samples per day.
| Product Name: | Arginase Activity Assay Kit (Colorimetric) (BA0016) |
| SKU: | BA0016 |
| Detection Method: | Colorimetric (OD 430 nm) |
| Detection Range: | 0.3 to 20 U/L (2 h reaction); detection limit 0.3 U/L |
| Sample Type: | ['Enzyme preparations', 'Serum', 'Plasma', 'Tissue culture', 'Cell lysates'] |
| Species Reactivity: | All |
| Assay Time: | ~3 hours (2 h reaction plus 60 min colour development) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Arginine Buffer and Urea Standard at -20°C; all other components at 2-8°C |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
Arginase (L-arginine ureohydrolase EC 3.5.3.1) is present in mammals and plants. In humans, arginase is expressed predominantly in the liver and to lesser degrees in breast, kidney, testes, salivary glands, epidermis and erythrocytes. Arginase catalyses the conversion of arginine to ornithine and urea, completing the last step in the urea cycle. Arginase activity is a key diagnostic indicator: increased levels in blood have been associated with liver damage, and hyperargininemia due to arginase deficiency is an inherited autosomal recessive disease. This kit provides a sensitive and convenient method for arginase activity determination using a chromogen that forms a coloured complex specifically with urea produced in the arginase reaction, with the intensity of the colour directly proportional to the arginase activity in the sample.
- Sensitive and accurate. Detection limit: 0.3 U/L for a 2 hour arginase reaction in the 96-well assay format.
- Simple and high-throughput. The procedure involves incubating the provided substrate with the sample in a microplate followed by addition of the colouring reagent, and can be readily automated as a high-throughput assay for thousands of samples per day.
- Direct Assays: arginase activity in enzyme preparations, serum, plasma, tissue culture etc.
- Drug Discovery/Pharmacology: effects of drugs on arginase activity.
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 | Prepare samples: serum and plasma contain urea, which should be depleted using a 10 kDa membrane filter by loading up to 100 µL sample, diluting to 500 µL with water, centrifuging at 14,000 rpm for 30 min and repeating; decant the concentrated sample and adjust the final volume. For cell lysates, harvest approximately 10^6 cells, wash with PBS, centrifuge at 1,000 g at 4°C for 10 min, lyse pellets for 10 min in 100 µL of 10 mM Tris-HCl (pH 7.4) containing 1 µM pepstatin A, 1 µM leupeptin and 0.4% Triton X-100, centrifuge at 14,000 g at 4°C for 10 min and use the supernatant. |
| 2 | Bring all reagents to room temperature, preheat the Arginine Buffer to 37°C and use reconstituted reagents within 2 hours. Prepare 1 mM Urea Standard by mixing 24 µL of 50 mg/dL urea and 176 µL water, then add 50 µL of 1 mM Urea Standard and 50 µL dH2O to separate wells of a 96-well plate. |
| 3 | Prepare 5x Substrate Buffer by combining 4 volumes of Arginine Buffer with 1 volume of Mn Solution (10 µL per test). Add 40 µL of each sample to two separate wells; add 10 µL of 5x Substrate Buffer to one sample well (ODSample) and leave the other without Substrate Buffer (Sample Blank Control, ODBlank). Incubate the reaction plate at 37°C for 2 hours or the desired reaction time. Dilute samples in water if necessary so apparent activities lie between 1 and 10 U/L. |
| 4 | Prepare Urea Reagent by combining equal volumes of Reagent A and Reagent B, add 200 µL to all wells (this stops the arginase reaction) and then add 10 µL of 5x Substrate Buffer to the Sample Blank Control well. Tap the plate to mix, incubate 60 min at room temperature and read the optical density at 430 nm. |
Arginase = [(ODSample - ODBlank) / (ODStandard - ODWater)] x [Urea Standard] x 50 x 10^3 / (40 x t), which simplifies to Arginase = [(ODSample - ODBlank) / (ODStandard - ODWater)] x 10.4 (U/L), where ODSample, ODBlank, ODStandard and ODWater are the optical density values of the sample, sample blank, standard and water respectively, [Urea Standard] = 1 mM, t is the reaction time (120 min) and 50 and 40 are the reaction and sample volumes in µL. If (ODSample - ODBlank)/(ODStandard - ODWater) is larger than 2, dilute the sample in distilled water and repeat multiplying by the dilution factor, or use a shorter reaction time. Unit definition: 1 unit of arginase converts 1 µmole of L-arginine to ornithine and urea per minute at pH 9.5 and 37°C.
| Component | Quantity | Storage |
| Arginine Buffer (pH 9.5) | 1.5 mL | -20°C |
| Mn Solution | 300 µL | 2-8°C |
| Reagent A | 12 mL | 2-8°C |
| Reagent B | 12 mL | 2-8°C |
| Urea standard (50 mg/dL) | 0.5 mL | -20°C |
