There are many ways to determine the concentration of protein solution. The following methods are available for selection. The determination of protein concentration is often an important method for calculating the recovery of a purification method.
First, the direct determination of protein concentration (UV method)
This method is to test the protein directly at a wavelength of 280 nm. Select the Warburg formula, the photometer can directly show the sample concentration, or select the appropriate conversion method to convert the absorbance value to the sample concentration. The protein determination process is very simple. Test the blank first and then test the protein directly. As a result, the result is very unstable. Protein direct quantification methods are suitable for testing relatively pure, relatively single-component proteins. UV direct quantification is faster and easier to perform than colorimetry; however, it is susceptible to interference from parallel substances, such as DNA interference, and low sensitivity requires high protein concentrations.
Second, colorimetric protein concentration determination of protein is usually a variety of protein compounds, colorimetry is based on the determination of protein components: amino acids (such as tyrosine, serine) and additional chromogenic groups or dye reaction, resulting in colored substance. The concentration of the colored material is directly related to the number of amino acids the protein reacts, thereby reflecting the protein concentration.
First, the biuret method The biuret method is the first method to measure protein concentration by colorimetry, and it is still widely used today. This method is commonly used in fast, but not very accurate, assays. The principle of the biuret method is the peptide bond between Cu2+ and the protein, and the complexation of tyrosine residues to form a purple-blue complex that has maximum absorption at 540 nm. The biuret method is usually used for the determination of a protein solution having a content of 0.5 g/L to 10 g/L. Interferers include thiols and peptide-based buffers such as Tris, Good buffers, and the like. The interference can be removed by precipitation by precipitating the protein with an equal volume of 10% cold trichloroacetic acid, discarding the supernatant, and quantifying the protein by dissolving the precipitated protein with a known volume of 1 M NaOH.
Three, BCA (Bicinchoninine acid assay) method
This is a newer, more sensitive method of protein testing. The protein to be analyzed reacts with Cu2+ in an alkaline solution to produce Cu+, which forms a chelate with BCA to form a purple compound with an absorption peak at 562 nm. The linear relationship between the concentration of this compound and the protein is extremely strong, and the compound formed after the reaction is very stable. Relative to the Lowry method, the operation is simple and the sensitivity is high. However, similar to the Lowry method, it is susceptible to interference with proteins and detergents.
Fourth, Lowry method
This method is a further development of the biuret method. His first step was the biuret reaction, in which Cu++ forms a complex with the protein in an alkaline solution, and this complex then reduces the phosphomolybdophosphoric-phosphotungstic acid reagent (Folin-phenol reagent), resulting in a dark blue. Color material. This method is much more sensitive than the biuret method and is suitable for the determination of a protein solution with a content of 20 mg/L to 400 mg/L. The interfering substance is the same as the biuret method. It should be noted that special care should be taken when adding the Folin reagent. The reagent is only stable in an acidic pH environment. The above-mentioned reduction reaction occurs only at pH 10, therefore, When the reagent is added to the alkaline Cu2+-protein solution, it must be stirred immediately so that the phosphomolybdic acid-phosphotungstic acid reagent can be effectively reduced by the Cu2+-protein complex before it is destroyed.
Reagents: 1. Reagent A: 2% Na2CO3/0.1 N NaOH. (Na2CO 310 g, NaOH 2 g diluted to 500 ml with distilled water).
2. Reagent B: Ba : 1% CuSO4B5H2OBb : 2% potassium sodium tartrate (prepared and stored separately in a brown bottle. Do not shake the pellet when used.)
3. Reagent C: Mix 0.1ml of Ba 0.1ml with 0.1ml of Bb, and add A 10ml of 4.Folin C Ciocalten reagent: dilute 1:1 with water before use. 5. BSA: Protein Standard Solution 1mg/ml: 10mg Crystalline calf serum protein is added to the surface of a 10ml volumetric flask of 5ml distilled water. After the solution is dissolved, do not shake and bubbling. Rinse to 10ml with distilled water.
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