Summary of common protein concentration determination methods

Summary of common protein concentration determination methods

Protein is one of the most important nitrogen-containing biological macromolecules in cells, and it bears various biological functions. Quantitative analysis of proteins is the basis for protein structural analysis. At present, the commonly used methods for protein measurement include BCA, Bradford and Lowry.

BCA method

principle

The protein is combined with the Cu2+ complex in an alkaline environment to reduce Cu2+ to Cu1+ (biuret reaction). BCA combines with Cu1+ to form a stable purple-blue complex with a high light absorption at 562 nm and is proportional to the protein concentration, from which the protein concentration can be determined.

Experimental procedure

1. Configure the BCA working fluid according to the kit instructions;

2. Completely dissolve the protein standard, add it to the well of the 96-well plate protein standard, and gradually dilute the standard according to the instructions, add the BCA working solution, and measure the absorbance with a microplate reader;

3. Draw a standard curve with the sample concentration as the X axis and the absorbance as the Y axis;

4. Add BCA working solution to the sample to be tested, measure the absorbance, bring it into the standard curve, and calculate the sample concentration.

Compared with the Lowery method, the BCA protein assay has high sensitivity and simple operation, and the reagents and the color complexes formed thereof are excellent in stability and are less affected by interfering substances. A significant advantage of the BCA method over the Bradford method is that it is not affected by detergents.

Coomassie Bradford Act

principle

The Coomassie brilliant blue G-250 assay for protein content is one of the dye binding methods. It is red in the free state, and the maximum light absorption is at 488 nm; when it binds to the protein, it turns cyan, and the protein-pigment conjugate has maximum light absorption at a wavelength of 595 nm. Its light absorption value is proportional to the protein content, so it can be used for the quantitative determination of protein, which is a commonly used rapid method for the determination of trace proteins.

Experimental procedure

1. Configure Bradford dye solution according to the kit instructions;

2. Completely dissolve the protein standard, add it to the well of the 96-well plate protein standard, and gradually dilute the standard according to the instructions. After adding the dye solution and the dye binding solution, measure the absorbance with a microplate reader;

3. Draw a standard curve with the concentration of the standard as the X-axis and the absorbance as the Y-axis;

4. Add dye solution and dye binding solution to each well, measure the absorbance of the sample, bring it into the standard curve, and calculate the sample concentration.

The Bradford reagent is simple to prepare, simple and quick to operate, and the reaction is very sensitive. The sensitivity is 4 times higher than the Lowry method. The microgram protein content can be determined. The protein concentration range is 0～1 000μg/mL, and the minimum detectable protein is 2.5μg/mL. .

Feilin-Phenol Reagent (Lowry)

principle

  The Folin-phenol reagent combines the reaction of a biuret reagent with a phenol reagent with a protein, including a two-step reaction: the first step is to react with a copper reagent to form a protein-copper complex under basic conditions. The second step is that the complex reduces the phosphomolybdic acid and phosphotungstic acid reagents to form a dark blue mixture of phosphorus molybdenum blue and phosphotungsten blue. The color depth is positively correlated with the protein content, and has maximum light absorption at a wavelength of 650 nm .

Experimental procedure

Much like the above two methods

The LORRY method has high sensitivity and good repeatability, and is suitable for quantification of 5 to 100 μg of protein. It takes a long time and requires strict timing. In the experiment, special attention should be paid to the interference of reducing substances and citric acid to obtain the most accurate experimental results.

The commonality of these methods is that the standard curve needs to be drawn, and the standard curve and the sample to be tested tend to be more. In order to shorten the time consumption of the experiment and ensure the measurement accuracy, the absorbance can be measured using a microplate reader.