Determination of protein content → biuret method

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Determination of protein content using the biuret method

Objective and Requirements

The aim of this experiment is to understand and apply the biuret method for determining protein concentration. This technique is widely used in biochemical analysis due to its simplicity and accuracy.

Experimental Principle

The biuret reaction occurs when compounds containing two or more peptide bonds are present. In an alkaline environment, biuret reacts with copper ions to form a violet-colored complex. Proteins and peptides also contain multiple peptide bonds, allowing them to react similarly with Cu²⁺ ions, forming a purple-red coordination compound. The absorbance of this complex at 540 nm is directly proportional to the protein concentration. This principle makes the biuret method ideal for quick and reliable protein quantification.

Reagents and Equipment

1. Reagent: Biuret reagent

Prepare 1.5 g of copper sulfate (CuSO₄·5H₂O) and 6.0 g of sodium potassium tartrate (NaKC₄H₄O₆·4H₂O) in 500 mL distilled water. Then add 300 mL of 10% NaOH solution while stirring. Dilute the mixture to 1000 mL with distilled water. Store the solution in a sealed container for long-term use.

2. Standard and Test Protein Solutions

(1) Standard protein solution: Prepare 10 mg/mL of crystalline bovine serum albumin (BSA) or casein solution using 0.05 mol/L NaOH. Ensure the standard solution is accurately prepared based on the known purity of the protein, determined previously by the micro-Kjeldahl method.

(2) Test protein solution: Use diluted human serum (1:10). For other samples, adjust the dilution factor so that the protein concentration falls within the linear range of the standard curve.

3. Equipment

Test tubes (1.5 × 15 cm, ×16), test tube rack, pipettes (1 mL ×3, 5 mL ×1), constant temperature water bath, and a spectrophotometer.

Precautions

(1) Perform colorimetric measurements within 30 minutes after the reaction has developed. Ensure consistent timing between each sample to maintain accuracy.

(2) If the sample contains a high level of lipids, the reaction mixture may become cloudy. In such cases, clarify the solution by adding ethanol or petroleum ether, followed by centrifugation. Measure the supernatant after clarification.