There are many methods for the determination of protein concentration. Coomassie Brilliant Blue is the most common method in the laboratory. It uses a colorimetric method and a pigment method to make it easy to operate. The principle of Coomassie Brilliant Blue determination of protein concentration is described below. Disadvantages, operations, and precautions.
Experimental principle The Coomassie Brilliant Blue method for the determination of protein concentration is a rapid and sensitive method for the quantitative determination of trace protein concentrations using the principle of protein-dye binding. This protein assay has outstanding advantages over several other methods and is now being widely used. This method is currently the most sensitive protein assay. Coomassie Brilliant Blue G-250 dye binds to protein in an acidic solution, changing the position of the maximum absorption peak (lmax) of the dye from 465 nm to 595 nm, and the color of the solution also changes from brownish black to blue. The amount of protein bound thereto was determined by measuring the amount of increase in light absorption at 595 nm. Studies have found that dyes are primarily combined with basic amino acids (especially arginine) and aromatic amino acid residues in proteins. The outstanding advantages of Coomassie blue staining are: (1) High sensitivity, estimated to be about four times higher than the Lowry method, and the minimum protein detection amount is up to 1 mg. This is because the color produced by the combination of protein and dye varies greatly, and the protein-dye complex has a higher extinction coefficient, so the light absorption value varies greatly with the protein concentration than the Lowry method. (2) The measurement is quick and simple, and only one reagent is added. It takes only about 5 minutes to complete the measurement of a sample. Since the dye-protein binding process is completed in about 2 minutes, the color can be stabilized within 1 hour, and the color stability is best between 5 minutes and 20 minutes. Therefore, it is not necessary to control the time in a time-consuming and strict manner as in the Lowry method. (3) Less interfering substances. Such as K+, Na+, Mg2+ ions, Tris buffer, sugar and sucrose, glycerol, mercaptoethanol, EDTA, etc., which interfere with the Lowry method, do not interfere with this assay. The disadvantages of this method are: (1) Due to the different contents of arginine and aromatic amino acids in various proteins, Coomassie blue staining is used for different protein determinations, which is usually used when making standard curves. G-globulin is selected as the standard protein to reduce the deviation in this respect. (2) There are still some substances that interfere with the determination of this method. The main interfering substances are: detergent, Triton X-100, sodium dodecyl sulfate (SDS) and so on. Reagents and equipment I. Reagent Coomassie Brilliant Blue reagent: Coomassie Brilliant Blue G-250 100 mg dissolved in 50 mL of 95% ethanol, added with 100 mL of 85% phosphoric acid, diluted to 1000 mL with distilled water. 2. Standard and protein solution to be tested 1. Standard protein solution crystallized bovine serum albumin. The protein nitrogen content was determined by micro-Kjeldahl method in advance, and 1 mg/mL protein solution was prepared according to its purity with 0.15 mol/L NaCl. 2. The protein solution to be tested. Human serum was diluted 200-fold with 0.15 mol/L NaCl before use. Third, equipment test tube 1.5 × 15cm (×6), test tube rack, pipette tube 0.5mL (×2); 1mL (×2); 5mL (×1); constant temperature water bath; spectrophotometer, US analysis UV-1100 ; UV-1200. Operation method 1. Make a standard curve and take 7 test tubes, and operate in parallel according to the following table. Tube No. 0 1 2 3 4 5 6 Standard protein solution (mL) 0 0.01 0.02 0.03 0.04 0.05 0.06 0.1 0.09 0.08 0.07 0.06 0.05 0.04 Coomassie Brilliant Blue Reagent (mL) 5 mL Shake well, with No. 0 tube as blank control within 1 h. A standard curve was drawn at 595 nm for colorimetric A595 nm: with A595nm as the ordinate and standard protein content as the abscissa, a standard curve was drawn on the coordinate paper. Second, the unknown sample protein concentration determination method is the same as above, take the appropriate unknown sample volume, so that the measured value is within the straight line range of the standard curve. Based on the measured A595nm value, the amount corresponding to the standard protein was found on the standard curve to calculate the protein concentration (mg/mL) of the unknown sample. Note Light absorption is measured within 5-20 minutes after the reagent is added because the color is most stable during this time. In the measurement, a small amount of the protein-dye complex is adsorbed on the wall of the cuvette, and the blue cuvette can be washed with ethanol after the measurement. The analysis of protein by Coomassie Brilliant Blue method must master the correct use of the spectrophotometer. When measuring the absorbance repeatedly, the cuvette must be rinsed clean. When making the protein standard curve, the protein standard is preferably from low to high. Determination to prevent errors. Key words: ultraviolet spectrophotometer; protein concentration; US instrumentation ; UV-1100; UV-1200The Cutting Machine is a product developed for Cutting TPU Protective Films, Hydrogel Screen Protective Films and PVC Back Stickers.
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