Determination of protein content → biuret method

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Determination of protein content → Biuret method

Purpose and Requirements

The objective of this experiment is to understand the principles and techniques involved in determining protein concentration using the biuret method. This method is widely used due to its simplicity and speed, making it suitable for routine laboratory analysis.

Experimental Principle

Compounds containing two or more peptide bonds exhibit a biuret reaction. When biuret reacts with copper ions in an alkaline environment, it forms a violet-colored complex. Proteins and peptides also contain multiple peptide bonds, which allow them to form a similar purple-red coordination compound with Cu²⁺. The absorbance of this complex reaches a maximum at 540 nm. The intensity of the color is directly proportional to the protein concentration, enabling quantitative analysis through spectrophotometry.

Reagents and Equipment

1. Reagent: Biuret Reagent

Prepare by dissolving 1.5 g of copper sulfate (CuSO₄·5H₂O) and 6.0 g of sodium potassium tartrate (NaKC₄H₄O₆·4H₂O) in 500 mL of distilled water. Add 300 mL of 10% NaOH solution while stirring, then dilute to 1000 mL with distilled water. This reagent is stable and can be stored for extended periods.

2. Standard Protein Solution and Sample Solution

(1) Standard Protein Solution: Prepare a 10 mg/mL solution of crystalline bovine serum albumin (BSA) or casein using 0.05 mol/L NaOH. The concentration of the standard should be accurately determined using the micro-Kjeldahl method, and the solution should be prepared based on its purity.

(2) Sample Protein Solution: Human serum diluted 10 times. For other samples, adjust the dilution factor so that the 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 the colorimetric measurement within 30 minutes after the reaction develops. Ensure that the time between color development and measurement is consistent across all tubes.

(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. Then measure the absorbance of the supernatant.