The Folin-phenol reagent method was the first to determine the basic steps of protein concentration determination by Lowry. Later it will be widely used in the field of biochemistry. The advantage of this method is its high sensitivity, which is much more sensitive than the biuret method. The disadvantage is that it takes a long time. It requires precise control of the operating time. The standard curve is not strictly linear, and it has poor specificity and many interfering substances . The Folin-phenol reagent method is also suitable for the quantitative determination of tyrosine and tryptophan. The determination range of this method is 20 ~ 250mg, and the minimum amount of protein detected can reach 5mg.

The color development principle of the Folin-phenol method is the same as the biuret method, except that the second reagent, the Folin-phenol reagent, is added to increase the amount of color development, thereby increasing the sensitivity of protein detection. Protein contains phenolic tyrosine. In alkaline solution, its peptide bond chelate with Cu2 + to form a protein-copper complex. This complex reduces the phosphomolybdic acid of the phenol reagent to produce a blue compound. Under certain conditions Next, use the linear relationship between the blue shade and the protein concentration to make a standard curve and determine the protein concentration in the sample.

Ions that interfere with the biuret reaction are also likely to interfere with the Lowry reaction. And the impact on the latter is much greater. Phenols, citric acid, ammonium sulfate, Tris buffer, glycine, sugars, glycerol, etc. all interfere with the Lowry reaction. Low concentration urea (0.5%), sodium sulfate (1%), sodium nitrate (1%), trichloroacetic acid (0.5%), ethanol (5%), ether (5%), acetone (0.5%), etc. The solution has no effect on the color development, but when the concentration of these substances is high, a calibration curve must be made. For the solution containing ammonium sulfate, it is only necessary to add concentrated sodium carbonate-sodium hydroxide solution to determine the color. If the acidity of the sample is high and the color will become light after color development, the concentration of the sodium carbonate-sodium hydroxide solution must be increased by 1 to 2 times.

In addition, be careful when adding Folin-phenol reagent, because the reagent is only stable under acidic pH conditions, but the above reduction reaction only occurs at pH = 10, so when Folin-phenol reagent is added to alkaline In the copper-protein solution, it must be mixed immediately so that the reduction reaction can occur before the phosphomolybdic acid-phosphotungstic acid reagent is destroyed.