The biuret method is an analytical technique used for protein quantification. The principle of the biuret reaction is that peptide bonds complex with copper ions in the presence of an alkaline copper sulfate solution (which is blue), generating a blue-violet compound. The absorbance of this compound at 540 nm is directly proportional to the number of peptide bonds, allowing for the calculation of protein content. The biuret method for protein concentration determination demonstrates good compatibility and is largely unaffected by other components in most samples, although it is susceptible to copper ion chelators. Additionally, for biuret analysis of serum total protein, bilirubin, lipids, hemoglobin, and dextran can cause some interference. The biuret method shows good linearity within the concentration range of 5–160 mg/mL. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
B1507239
| Component
| 500T | 1000T | Storage
|
B1507239A
| Biuret Reagent
| 100 mL | 200 mL | RT
|
B1507239B
| Protein Standard (BSA)
| 40 mg | 40 mg
| RT
|
B1507239C
| Protein Standard Diluent
| 1.5 mL | 3 mL | RT |
Materials Required but Not Provided
Test samples (serum, plasma, tissue homogenate)
Centrifuge tubes or small test tubes, water bath or incubator, microplate reader or spectrophotometer, 96-well plate or cuvettes
Physiological saline (0.9% NaCl) or PBS
Experimental Procedure
1. Preparation of Standard
Add 0.25 mL of Protein Standard Diluent (or an appropriate diluent) to the 40 mg Protein Standard (BSA) vial. Dissolve completely to prepare the Protein Standard Solution (160 mg/mL). This solution can be used immediately after preparation. The dissolved Protein Standard Solution should be stored at -20°C. It can also be diluted as needed, for example to 40 mg/mL.
Special Note: The protein standard should ideally be dissolved/diluted in the same solution as the protein in the test samples. For instance, if the test protein is dissolved in a sucrose solution, the protein standard should also be dissolved in sucrose solution. Commonly, 0.9% NaCl or PBS is used as the diluent for the total protein standard.
2. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard solution to the standard wells of a 96-well plate, respectively. Bring the volume in each standard well to 20 μL using the diluent.
3. Add an appropriate volume of the test protein sample to the sample wells of the 96-well plate.
If the standard diluent is different from the solution used to dissolve the test protein sample, add 20 μL of the diluent to the test sample wells.
If the standard diluent is the same as the solution used to dissolve the test protein sample, it is not necessary to add 20 μL of diluent to the test sample wells.
4. Add 200 μL of Biuret Reagent to each well. Incubate at room temperature for 10–15 minutes.
5. Measure the absorbance at 540 nm. If 540 nm is unavailable, wavelengths between 510–562 nm can also be used.
6. Calculate the protein concentration of the samples based on the standard curve.
Precautions
Dissolving the Protein Standard powder in the Protein Standard Diluent yields the stock Protein Standard Solution. This stock contains preservatives which do not interfere with the assay and can be stored long-term at -20°C.
If detection performance is suboptimal after adding Biuret Reagent to the test protein and standard, letting the reaction proceed at room temperature for 1 hour or at 60°C for 15 minutes may help, as the color continues to develop over time.
If no significant increase in absorbance or color is observed with increasing standard concentration during standard curve generation, the sample might contain copper ion chelators.
At protein concentrations below 20 mg/mL, the color reaction is a faint blue. A distinct blue-violet reaction becomes apparent at concentrations greater than 40 mg/mL.
The linear range of this kit is 5–160 mg/mL. Sample protein concentration should be greater than 1 mg/mL, preferably above 20 mg/mL. For low-concentration protein samples (~1 mg/mL), the Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.
The absorbance of the blue-violet compound generated by this kit depends on the reagent batch, pH, and reaction temperature. Therefore, it is recommended to run a standard control with each assay.
Amino acids and dipeptides do not yield the biuret reaction. The biuret complexes of tripeptides, oligopeptides, and polypeptides with copper ions are pink to purplish-red, differing from the protein biuret reaction result.
If a microplate reader is unavailable, a spectrophotometer can also be used. Using a spectrophotometer significantly reduces the number of samples measurable per kit.
If all wells appear dark purple, the sample might contain reducing agents. Consider appropriate dialysis or dilution of the sample.
Use reagents promptly after opening to prevent potential effects on subsequent experimental results.
The prepared Protein Standard Solution should be stored frozen at -20°C.
Appendix: Reference Standard Curve Range
The absorbance (un-zeroed) of protein standards at 1, 5, 10, 20, 40, 60, 80, 100, 120, 140, 160 mg/mL was measured at 540 nm using a microplate reader. After zeroing with the blank, the absorbance for 40 mg/mL typically ranged from 0.15 to 0.18. The resulting standard curve is shown below:
The results show that the absorbance for 1 mg/mL is relatively low and close to the blank. Therefore, it is recommended that the sample concentration for assay be greater than 1 mg/mL, preferably above 20 mg/mL. For low-concentration protein samples (~1 mg/mL), please use the Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit.
Note: For protein samples in the range of 8–16 mg/mL, fully consider potential interfering factors such as EDTA, 2-ME, and DTT. Detection results for these samples may fluctuate significantly, and the standard itself might also show variability. Please perform operations carefully and precisely.
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