Protein Content Assay Kit (Biuret, Micro Method)

Cat. No.: T1507240

GRADE & PURITY BioReagent, for Protein analysis

Overview

Total Protein (TP) consists of albumin and globulin. The determination of total protein content in biological fluids (serum, urine, cerebrospinal fluid) is generally based on two assumptions: 1) All protein molecules are composed of pure polypeptides, with a mass percentage of nitrogen content of 16%; 2) The body fluid contains hundreds of protein molecules, each exhibiting very similar characteristics in the assay reaction. Commonly used methods for total protein detection include the biuret method, UV spectrophotometry, dye-binding methods, the Kjeldahl method, and precipitation methods.

This product is primarily used for determining total protein content in human or animal serum, plasma, tissues, and other samples. 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 also shows good compatibility and is largely unaffected by other components in most samples, although it can be 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 demonstrates 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.

T1507240	Component	120T	Storage
T1507240A	Biuret Reagent	25 mL	2-8℃
T1507240B	Protein Standard	40 mg	RT
T1507240C	Protein Standard Diluent	2 mL	RT
T1507240D	Biuret Blank Reagent	10 mL	2-8℃

Materials Required but Not Provided

1. Test samples (serum, plasma, tissue), physiological saline (0.9% NaCl) or PBS

2. Centrifuge tubes or small test tubes, water bath or incubator, microplate reader, 96-well plate, homogenizer or mortar and pestle

Experimental Procedure

1. Preparation of Standard

Add 0.25 mL of Protein Standard Diluent (or an appropriate diluent) to the 40 mg Protein Standard 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. Sample Preparation

2.1 Serum/Plasma Samples: Use 4 μL directly for detection.

2.2 Tissue Samples: Add 9 volumes of physiological saline per mass of tissue (g) (e.g., 1 g tissue + 9 mL saline). Homogenize in an ice bath, then centrifuge at 2500 × g for 10 minutes. Collect the supernatant and adjust the volume to 10 mL with physiological saline for testing.

3. TP Assay Setup

Set up Blank, Standard, and Test wells according to the table below. Add reagents in the specified order, avoiding bubble formation. If the sample concentration is too high, reduce the sample volume or dilute appropriately before assay. Running sample duplicates is recommended.

Reagent (μL)	Blank Well	Standard Well	Test Well
ddH₂O	4	－	－
Protein Standard Solution (e.g., 40 mg/mL)	－	4	－
Test Sample	－	－	4
Biuret Reagent	200	200	200

Note: For turbid or hemolyzed samples, set up a "Sample Blank Well": Add 0.04 mL of test sample to 2 mL of Biuret Blank Reagent – this is the "Sample Blank Well". Zero the instrument with the Biuret Blank Reagent, then read the absorbance of the "Sample Blank Well". Use the net absorbance (A_test - A_{sample blank}) to calculate the total protein concentration.

4. TP Measurement

Mix well and incubate at 37°C for 10 minutes. Measure the absorbance of each well at 540 nm using a microplate reader, recorded as A_standard and A_test. If 540 nm is unavailable, wavelengths between 510–562 nm can also be used. Zero the instrument using the Blank well.

5. Calculation of Results

• Serum/Plasma Sample Total Protein Concentration (g/L) = (A_test/ A_standard) × C

• Turbid/Hemolyzed Sample Total Protein Concentration (g/L) = [(A_test - A_{sample blank}) / A_standard] × C

• Total Protein Content per 100g Tissue (g/100g) = (A_test / A_standard) × C × V_T× 100 / m

Parameter Description:

• A_test: Absorbance of the test well

• A_standard: Absorbance of the standard well

• A_{sample blank}: Absorbance of the sample blank well

• C: Protein Standard Solution concentration (mg/mL or g/L)

• V_T: Total volume of tissue supernatant = 10 mL

• m: Mass of the tissue sample (g)

• 100: Mass for reporting content per 100g of tissue

Reference Interval:

Condition	Serum Total Protein Concentration
Healthy ambulatory adults	64 ~ 83 g/L
Healthy recumbent adults	60 ~ 78 g/L

Precautions

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. A spectrophotometer can be used if a microplate reader is unavailable, but the number of samples measurable per kit will be significantly reduced.

9. If all wells appear dark purple, the sample might contain reducing agents. Consider appropriate dialysis or dilution of the sample.

10. Use reagents promptly after opening to prevent potential effects on subsequent experimental results.

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.

Storage and Shipping

Storage

Store at 2-8°C,Room temperature

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.