BioReagent BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
Store at 2-8°C Ships Wet ice Check lot-specific COA for exact specifications.
SDS, COA, datasheet, and spec sheet available for download. Lot-specific COA accessible via lot number lookup.
Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
Under heating and strong acid conditions, urea reacts with diacetyl monoxime and antipyrine to form a yellow compound, which has a characteristic absorption peak at 460 nm. By measuring the increase in absorbance of the yellow compound at 460 nm, the urea content in the sample can be calculated.
Reagents, consumables and Equipments not provided
Microplate reader (capable of measuring absorbance at 460 nm)
Mortar (homogenizer), ice box (ice machine), benchtop centrifuge, water bath (oven, incubator, metal bath)
Adjustable pipettes, 96-well plates, centrifuge tubes
Distilled water (deionized water / ultrapure water are both acceptable)
Procedure (For Reference Only)
It is recommended to first select 1-3 samples with significant differences (e.g., different types/grouped samples) for a pilot experiment to familiarize yourself with the procedure. Based on the pilot experiment results, determine/adjust the sample dilution factor to avoid wasting samples or reagents.
1. Reagent Preparation
| Reagent | Instructions |
|---|---|
| Reagent A | 1. Before opening, tap the tube to ensure the powder settles at the bottom (can be done manually). 2. Add 3 mL of distilled water and dissolve completely. 3. After dissolution, the storage period is consistent with the kit's expiry date. |
| Reagent B | 1. Before opening, tap the tube to ensure the powder settles at the bottom (can be done manually). 2. First, add 2.4 mL of distilled water to dissolve. 3. Slowly add 2.4 mL of phosphoric acid and mix thoroughly. 4. Then add 4.8 mL of sulfuric acid, mix well, and cool to room temperature before use. 5. After preparation, the storage period is consistent with the kit's expiry date. |
| Standard Tube | For each standard tube, proceed as follows: 1. Before use, add 1 mL of distilled water to dissolve (concentration = 4 mg/mL). 2. Dilute 40-fold with distilled water at a 1:39 ratio to prepare a 0.1 mg/mL standard solution. 3. After dilution, the storage period is consistent with the kit's expiry date. |
2. Sample Preparation
2.1 Tissue Samples
Take 0.1 g of tissue sample and add 1 mL of distilled water. Homogenize.
Perform ultrasonic extraction for 10 min, then adjust the volume back to 1 mL. Let stand at room temperature for 30 min.
Centrifuge at 12,000 rpm at room temperature for 10 min. Collect the supernatant for testing.
2.2 Liquid Samples
Clear liquid samples can be tested directly.
If the sample is turbid, centrifuge and collect the supernatant for testing.
3. Assay Procedure
3.1 Pre-heat the microplate reader for 30 min, set the temperature to 37°C, and the detection wavelength to 460 nm.
3.2 Before the experiment, select 2 samples to determine the appropriate dilution factor (D) for this assay.
3.3 Thaw all reagents to room temperature. Add the following volumes (in µL) to EP tubes:
| Reagent Component | Test Tube | Control Tube | Blank Tube (Perform once) | Standard Tube (Perform once) |
|---|---|---|---|---|
| Sample | 50 | 50 | — | — |
| Distilled Water | — | 20 | 50 | — |
| Standard Solution | — | — | — | 50 |
| Reagent A | 20 | — | 20 | 20 |
| Reagent B | 40 | 40 | 40 | 40 |
3.4 Mix thoroughly. Incubate in a 95°C boiling water bath for 20 min.
3.5 Add 190 µL of distilled water to each tube and mix again. If the solution becomes turbid, centrifuge at 5,000 rpm at room temperature for 5 min and collect the supernatant.
3.6 Transfer 200 µL of the above solution to a 96-well plate. Measure the absorbance (A) at 460 nm. Calculate ∆A = A<sub>Test</sub> - A<sub>Control</sub>.
【Note】 If the absorbance (A) of the Test Tube exceeds 1.5, further dilute the sample and incorporate the final dilution factor (D) into the subsequent calculation formula.
4. Calculation of Results
4.1 Calculation Based on Tissue Weight
Urea Content (mg/g tissue) = (CStandard × V1) × ∆A ÷ (AStandard - ABlank) ÷ (V1 ÷ V × W) × D
= 0.1 × ∆A ÷ (AStandard - ABlank) ÷ W × D
Urea Content (mg/kg tissue) = (CStandard × V1) × 10³ × ∆A ÷ (AStandard - ABlank) ÷ (V1 ÷ V × W) × D
= 100 × ∆A ÷ (AStandard - ABlank) ÷ W × D
Urea Nitrogen Content (µg/g tissue) = (CStandard × V1) × ∆A ÷ (AStandard - ABlank) ÷ (V1 ÷ V × W) × D × 10³ ÷ 60.04 × 2 × 14
= 46.64 × ∆A ÷ (AStandard - ABlank) ÷ W × D
4.2 Calculation Based on Liquid Volume
Urea Content (mg/mL) = (CStandard × V1) × ∆A ÷ (AStandard - ABlank) ÷ V1 × D
= 0.1 × ∆A ÷ (AStandard - ABlank) × D
Urea Nitrogen Content (mg/dL) = (CStandard × V1) × ∆A ÷ (AStandard - ABlank) ÷ V1 × D × 100 ÷ 60.04 × 2 × 14
= 4.664 × ∆A ÷ (AStandard - ABlank) × D
4.3 Calculation Based on Protein Concentration
Urea Content (mg/mg prot) = (CStandard × V1) × ∆A ÷ (AStandard - ABlank) ÷ (V1 ÷ V × Cpr) × D
= 0.1 × ∆A ÷ (AStandard - ABlank) ÷ Cpr × D
Urea Content (mg/g prot) = (CStandard × V1) × 10³ × ∆A ÷ (AStandard - ABlank) ÷ (V1 ÷ V × Cpr) × D
= 100 × ∆A ÷ (AStandard - ABlank) ÷ Cpr × D
Urea Nitrogen Content (µg/mg prot) = (CStandard × V1) × ∆A ÷ (AStandard - ABlank) ÷ (V1 ÷ V × Cpr) × D × 10³ ÷ 60.04 × 2 × 14
= 46.64 × ∆A ÷ (AStandard - ABlank) ÷ Cpr × D
Parameter Description for Formulas
C<sub>Standard</sub>: Concentration of the urea standard, 0.1 mg/mL.
D: Sample dilution factor. D=1 if the sample is undiluted.
60.04: Molecular weight of urea.
2: One molecule of urea contains two nitrogen atoms.
14: Atomic weight of nitrogen.
W: Weight of the tissue sample taken (g).
V: Total volume of the sample extract (1 mL).
V<sub>1</sub>: Volume of the sample added to the reaction system (0.05 mL).
C<sub>pr</sub>: Protein concentration of the sample (mg/mL). It is recommended to use Aladdin's B665595 BCA Protein Assay Kit or R1491648 Ready-to-Use BCA Protein Assay Kit.
Precautions
Before formal testing, it is recommended to select 2-3 samples with expected significant differences for a pilot experiment.
For tissue samples and cell samples, results can be normalized between samples by measuring the protein concentration. It is recommended to use Aladdin's B665595 BCA Protein Assay Kit or R1491648 Ready-to-Use BCA Protein Assay Kit.
Biochemical detection reagents are generally irritating and have biological toxicity. For your safety and health, please ensure proper biosafety protection throughout the experiment. Wear a lab coat, mask, gloves, head cover, and other safety gear. Conduct experiments in a fume hood or biosafety cabinet.
This product is for scientific research only and is not intended for clinical diagnosis.
| U1516000 | Component | 96T | Storage |
| U1516000A | Reagent A | 1 EA | 2-8℃. |
| U1516000B | Reagent B | 1 EA | 2-8℃. |
| U1516000C | Standard Tube | 1 EA×2 | 2-8℃. |
Comprehensive hazard, handling, storage, and regulatory compliance document.
Download SDS →Lot-specific quality data. Enter your lot number to retrieve the exact COA.
Look up COA →Full quality attributes and acceptance criteria for this grade.
View spec sheet →Find and download the COA for your product by matching the lot number on the packaging.
| Lot Number | Certificate Type | Date | Item |
|---|---|---|---|
 ZJ26F0434006 | Certificate of Analysis | Apr 09, 2026 | U1516000 |
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