BioReagent BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
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The presence of protein in urine is termed proteinuria. While normal urine contains minimal trace amounts of protein, qualitative tests under healthy conditions should yield a negative result. Persistent elevation of urinary protein levels, indicated by a positive qualitative test, often signifies renal pathology or impairment. Consequently, changes in urinary protein concentration are a critical parameter for diagnosing kidney disease and assessing the extent of damage.
Detection Principle
Ponceau S dye binds to proteins in the urine sample, forming an insoluble protein-dye complex. After centrifugation, the supernatant is discarded, and the pellet is dissolved in an alkaline buffer. This dissociates the complex, restoring the dye's colorimetric properties. The absorbance is measured at 560 nm using a spectrophotometer. The absorbance value is proportional to the protein concentration, allowing for the calculation of protein content in the sample by comparison with a standard curve. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
| H1506744 | Component | 100T | Storage |
| H1506744A | Sulfosalicylic Acid Solution | 100 mL | RT. Store in the dark. |
| H1506744B | Ponceau S Reagent (10X) | 10 mL | RT. Store in the dark. |
| H1506744C | Alkaline Buffer | 200 mL | RT. |
| H1506744D | Protein Standard (BSA) | 20 mg | RT. |
| H1506744E | Protein Standard Diluent | 1.5 mL | RT. |
Materials Required but Not Provided
Distilled water
Centrifuge tubes, test tubes, cuvettes, spectrophotometer
Experimental Procedure
1. Preparation of 1X Ponceau S Working Solution
Dilute an appropriate amount of the Ponceau S Reagent (10X) with distilled water at a 1:9 ratio (e.g., 1 mL of 10X Reagent + 9 mL water). Mix well. This working solution can be stored at room temperature protected from light and remains stable for 2-3 months.
2. Preparation of Protein Standard Stock Solution (20 mg/mL)
Add 1 mL of Protein Standard Diluent to the vial containing the Protein Standard (BSA). Dissolve completely to prepare a 20 mg/mL protein standard stock solution. The dissolved stock solution can be used immediately and should be stored at -20°C for long-term preservation.
3. Preparation of Diluted Protein Standard Solutions
Dilute the 20 mg/mL Protein Standard Stock to concentrations such as 0, 200, 400, 600, 800, 1000, 1200, 1600 μg/mL, or other desired concentrations.
Note: To maintain matrix consistency, the solution used for diluting the standards should ideally match the matrix of the test urine samples. For instance, if the target protein is dissolved in sucrose, the 20 mg/mL standard should also be diluted in sucrose. Generally, 0.9% NaCl or PBS can also be used to dilute the BSA standard. Diluted protein standards should also be stored at -20°C.
IV. Generating the Protein Standard Curve
Take 100 μL of each diluted protein standard and process them identically to the test samples (see Step 6). Use the "0" concentration standard as the blank to zero the instrument. Measure the absorbance at 560 nm using a cuvette with a 1 cm path length. Plot the standard curve using concentration vs. absorbance.
4. Semi-Quantitative Preliminary Test
4.1 Place 3-5 mL of fresh urine in a small test tube.
4.2 Slowly add 2-3 drops of Sulfosalicylic Acid Solution down the inner wall of the tube to form a layer on top of the urine.
4.3 Immediately observe the interface against a black background. Estimate the approximate protein concentration range based on the following table:
| Result Interpretation | Phenomenon Description | Estimated Protein Concentration |
| Negative (-) | No turbidity, clear and transparent | < 0.05 g/L |
| Suspicious (±) | Slight turbidity, faintly visible | 0.05 - 0.2 g/L |
| (+) | Distinct white turbidity, no granules | ~ 0.3 g/L |
| (2+) | Thin, milky turbidity with appearing granules | ~ 1 g/L |
| (3+) | Milky and opaque, with flocculent precipitates | ~ 3 g/L |
| (4+) | Flocculent precipitates, coagulated into clumps | > 5 g/L |
6. Quantitative Detection
6.1 Determining Sample Volume
Based on the result of the semi-quantitative test, select an appropriate sample volume for detection according to the table below:
| Estimated Protein Concentration | Sample Volume | Dilution Factor | |
| < 1 g/L | 100 μL | 0 μL | 1 |
| 1 - 3 g/L | 50 μL | 50 μL | 2 |
| > 3 g/L | 10 μL | 90 μL | 10 |
6.2 Precipitation Reaction
Add the determined sample volume and distilled water (if any) to a centrifuge tube. Add 1 mL of 1X Ponceau S Working Solution. Vortex to mix thoroughly. Centrifuge at 3500 × g for 10 minutes.
6.3 Washing and Dissolution
After centrifugation, carefully decant the supernatant. Invert the tube on absorbent paper for several minutes to drain. Use a filter paper strip to wipe away any excess reagent from the tube walls (avoid touching the pellet). Add 2 mL of Alkaline Buffer to the pellet and mix thoroughly to dissolve the precipitate.
6.4 Colorimetric Measurement
Using a spectrophotometer zeroed with distilled water, measure the absorbance of all standard and sample solutions at 560 nm (540-595 nm is also acceptable).
7. Calculation of Results
7.1 Plot the standard curve using the concentrations (μg/mL) of the diluted protein standards as the x-axis and their corresponding absorbance values as the y-axis. Obtain the regression equation.
7.2 Substitute the sample's absorbance value into the regression equation to calculate the measured concentration (C, in μg/mL).
7.3 Calculate the protein concentration in the original urine sample based on the dilution factor used during the assay:
Original Urine Protein Concentration (g/L) = [C (μg/mL) × Dilution Factor] / 1000
Reference Interval (in patients with kidney disease): 46.5 ± 18.1 mg/L
Precautions
The Protein Standard (BSA) powder dissolved in the Protein Standard Diluent yields the protein standard stock solution. This stock contains preservatives which do not interfere with the assay. This stock solution can be stored long-term at -20°C.
The protein standards should ideally be dissolved/diluted in the same type of solution as the protein in the test samples. Inconsistent non-protein components between the sample and standard matrices may lead to inaccurate results.
If the estimated protein concentration in the test sample is < 0.1 g/L, use 1 mL of sample mixed directly with 0.1 mL of Ponceau S Reagent (10X). Mix well and proceed with the remaining steps.
This kit is also compatible with detection using a microplate reader.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Use reagents promptly after opening to prevent potential effects on subsequent experimental results.
Comprehensive hazard, handling, storage, and regulatory compliance document.
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