Folin-Ciocalteu method for determining protein content standard curve
The Folin-Ciocalteu method, also known as the Lowry method, is a commonly used biochemical assay for determining total protein concentration. This method was proposed by Oliver H. Lowry and others in 1951. It is based on the reaction between protein, phenol, and copper ions that produces a complex which absorbs light at a specific wavelength, allowing the calculation of protein concentration.
The principle of the Folin-Ciocalteu method is that the aromatic peptide bonds and phenolic hydroxyl groups in proteins form a colored complex with copper ions. The absorbance of this complex at 750 nm is proportional to the protein concentration. This is an analytical method based on the Beer-Lambert law.
Preparation of Standard Curve
In the laboratory, we typically use a protein solution of known concentration (such as bovine serum albumin, BSA) as a standard to prepare a series of standard solutions of different concentrations. We then measure their absorbance to create a standard curve.
1. Dilute the known concentration of BSA solution proportionally to prepare a series of standard solutions. The concentrations are usually chosen within the experimental measurement range, such as 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/ml, etc.
2. Mix each standard solution with the Folin-Ciocalteu reagent and allow it to react uniformly for a period of time.
3. Measure the absorbance of each standard solution at 750 nm.
4. Plot the standard curve with absorbance on the vertical axis and concentration on the horizontal axis.
In experiments, we generally assume a linear relationship between absorbance and concentration. However, at very high or low concentrations, this relationship may deviate from linearity. Therefore, selecting an appropriate concentration range is essential to ensure measurement accuracy. The Folin-Ciocalteu method is a common and important method for determining total protein concentration. By creating a standard curve, we can accurately and quickly determine the concentration of unknown protein solutions. This method has wide applications in biochemistry, molecular biology, immunology, and other fields.
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