Description
Urea Assay Kit III (BA0190) (BA0190)
The Urea Assay Kit III (SKU: BA0190) provides a simple, direct and automation-ready colorimetric method for the quantitative determination of urea or blood urea nitrogen (BUN) in biological samples. Urea, the major end product of protein catabolism in animals, is produced primarily in the liver and secreted by the kidneys, and its determination is very useful for assessing kidney function; increased levels are associated with conditions such as nephritis and urinary tract obstruction, while decreased levels may indicate acute hepatic insufficiency. In this assay urease converts urea to ammonia and carbon dioxide, and NADH is then converted to NAD+ in the presence of ammonia, alpha-ketoglutarate and glutamate dehydrogenase; the resulting decrease in optical density at 340 nm is directly proportional to the urea concentration in the sample. With a linear detection range of 50 to 1000 uM using a 20 uL sample and a simple room-temperature procedure read after 30 minutes, the homogeneous format can be readily automated. It is intended for research use only.
| Product Name: | Urea Assay Kit III (BA0190) |
| SKU: | BA0190 |
| Detection Method: | Colorimetric (OD340 nm) |
| Detection Range: | 50 to 1000 uM urea in 96-well plate assay (20 uL sample) |
| Sample Type: | Plasma, serum, urine, bronchoalveolar lavage and food/beverage samples such as milk |
| Species Reactivity: | All |
| Assay Time: | 30 min |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt (3 weeks after reconstitution of dried NADH Reagent) |
| Shipping: | Gel Pack |
A colorimetric assay for the quantitative determination of urea or blood urea nitrogen. Urease converts urea to ammonia, and NADH is then converted to NAD+ in the presence of ammonia, alpha-ketoglutarate and glutamate dehydrogenase; the decrease in optical density at 340 nm is directly proportional to urea concentration. The linear detection range is 50 to 1000 uM (20 uL sample) and the room-temperature assay is read after 30 minutes.
- Fast and sensitive; linear detection range 50 to 1000 uM urea (20 uL sample)
- Convenient single-working-reagent procedure read after 30 minutes at room temperature
- No 37C heater is needed
- Homogeneous mix-incubate-measure format readily automated on high-throughput systems
- Determination of urea in biological samples such as plasma, serum, urine and bronchoalveolar lavage
- Determination of urea in food and beverage samples such as milk
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, briefly centrifuge tubes and reconstitute the NADH Reagent with 1 mL dH2O (final 10 mM). Prepare a 1000 uM Urea Standard Premix by mixing 15 uL of the 40 mM Standard with 585 uL dH2O and dilute as shown in the table. |
| 2 | Transfer 20 uL standards into separate wells of a clear flat-bottom 96-well plate. Transfer 20 uL of each sample into two separate wells, one as a Sample Blank (R_BLANK) and one as a Sample (R_SAMPLE). |
| 3 | Prepare Working Reagent per standard and sample well by mixing 180 uL Assay Buffer, 1 uL Enzyme, 8 uL reconstituted NADH Reagent, 1 uL Urease and 1 uL Ketoglutarate. Prepare blank control reagent by mixing 180 uL Assay Buffer, 8 uL reconstituted NADH Reagent, 1 uL Enzyme and 1 uL Ketoglutarate (no Urease). |
| 4 | Add 180 uL Working Reagent to the four Standards and the Sample wells, and 180 uL Blank Control Reagent only to the Sample Blank wells. Tap to mix and incubate 30 minutes at room temperature. |
| 5 | Read OD340nm. |
Subtract the standard values from the blank value (#4) and plot delta OD against standard concentrations to determine the slope. [Urea] = (ODBLANK - ODSAMPLE) / Slope x n (uM), where ODSAMPLE and ODBLANK are the readings of the Sample and Sample Blank respectively and n is the dilution factor. If the calculated urea concentration exceeds 1000 uM, dilute in dH2O and repeat, multiplying by the dilution factor n. Conversions: 1000 uM urea equals 6 mg/dL or 60 ppm; Urea BUN (mg/dL) = [Urea] (mg/dL) / 2.14.
| Component | Quantity | Storage |
| Assay Buffer | 20 mL | -20C |
| Enzyme | 120 uL | -20C |
| Ketoglutarate | 120 uL | -20C |
| Urease | 120 uL | -20C |
| NADH Reagent | Dried | -20C |
| Standard | 400 uL | -20C |
