What are the commonly used methods for detecting protein concentration?
In an alkaline environment, the protein complexed with Cu2 + to reduce Cu2 + to Cu1 + (biuret reaction). BCA combines with Cu1 + to form a stable purple-blue complex, which has a high light absorption value at 562nm and is proportional to the protein concentration.
- Prepare BCA working solution according to the instructions of the kit;
- Dissolve the protein standard sample completely, add it into the protein standard hole of 96 well plate, and carry out gradient dilution according to the requirements of the manual, add BCA working solution, and measure its absorbance with enzyme reader;
- Draw standard curve with sample concentration as X axis and absorbance as y axis;
- Add BCA working solution into the sample to be tested, and determine the absorbance The concentration of the sample is calculated.
Compared with Lowery method, BCA protein determination method has high sensitivity, simple operation, good stability of reagent and color complex formed, and less affected by interfering substances. Compared with Bradford method, BCA method is not affected by detergents.
Coomassie brilliant blue G-250 (Coomassie brilliant blue G-250) 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 combines with protein, it turns blue, and the protein pigment conjugate has the maximum light absorption at 595 nm. Its light absorption value is directly proportional to the protein content, so it can be used for the quantitative determination of protein. It is a common method for the rapid determination of trace protein.
- The Bradford dye solution was prepared according to the instructions of the kit;
- The protein standard was dissolved completely and added into the protein standard hole of the 96 well plates, and the standard was diluted gradient according to the requirements of the manual. After the dye solution and dye-binding solution were added, the absorbance was determined by an enzyme label instrument;
- The standard curve was drawn with the standard concentration as X-axis and the absorbance as Y-axis;
- Add dye solution and dye-binding solution into each hole, measure the absorbance of the sample, bring it into the standard curve, and calculate the sample concentration.
Bradford method has the advantages of simple preparation, simple operation, very sensitive reaction. The sensitivity of Bradford method is 4 times higher than Lowry method. It can determine the content of microgram protein. The concentration range of protein is 0-1 000 μ g / ml, and the minimum is 2.5 μ g / ml.
The Folin phenol reagent method combines the reaction of biuret reagent and phenol reagent with protein, which includes two steps: the first step is to form protein copper complex with copper reagent in alkaline condition; The second step is to reduce phosphomolybdic acid and phosphotungstic acid reagent to form a dark blue mixture of phosphomolybdate blue and phosphotungstic acid. The color depth is positively related to the protein content and has the maximum light absorption at 650 nm.
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