First, the Bradford method
The protein combines with the Coomassie brilliant blue dye to form a blue substance, and the amount of the blue substance is proportional to the level of the protein. The resulting blue substance had maximum light absorption at 595 nm, and the protein concentration was determined by measuring light absorption at 595 nm. Bovine serum albumin (BSA) is generally used as a standard curve in the experiment. The sensitivity of this method is 25~200ug/ml. Glycerin, tween 80, detergent, 2-mercaptoethanol, acetic acid, ammonium sulphate, Tris, and some alkaline buffers can interfere with the assay and can be set up by appropriate controls. To eliminate this interference.
Reagent preparation:
1. Bradford stock solution: 95% ethanol, 100mL; 88% phosphoric acid, 200mL; Coomassie brilliant blue G-250 dye, 350mg; stable storage at room temperature for a long time.
2. Bradford working solution: double distilled water, 425mL; 95% ethanol, 15mL; 88% phosphoric acid, 30mL; Bradford stock solution, 30mL; Whatman I filter paper filter, stored in a brown bottle at room temperature, available for several weeks, but need to be re-used before use filter.
3. Standard protein: 1 mg/mL bovine serum albumin.
Operation method:
1. Using PBS as a blank control, standard protein BSA gradients were diluted to 20 ug/mL, 17.5 ug/mL, 15 ug/mL, 12.5 ug/mL, 10 ug/mL, 7.5 ug/mL, 5 ug/mL, 2.5 ug/ mL, the sample to be tested is properly diluted, 100 uL per tube. A new standard curve should be made for each experiment; there are no samples of 2~3 repeat tubes.
2. Add 1 mL of Bradford working solution, mix by shaking, and react at room temperature for 2 minutes. Since the reaction between the dye and the protein is continuous, the dye should be added to the protein sample in turn, and all samples should also be tested for the A595 value in the order in which the dye is added.
3. The A595 value is determined and the measurement should be completed within 1 hour.
4. The concentration of the standard protein is plotted on the abscissa, and the A595 value is plotted on the ordinate. The concentration is determined by the A595 value of the sample to be tested according to the standard curve.
Second, the Lowry method
The Lowry method, also known as the Folin-phenol method, forms a copper-protein complex with an appropriate reagent in an alkaline solution. This complex reduces the Folin-phenol reagent (phosphomolybdic acid-phosphoric acid reagent) to produce a deep blue molybdenum. Blue and tungsten blue complex, this dark blue complex has maximum light absorption at 745~750nm, and the absorbance is proportional to the protein concentration.
Reagent preparation:
1. Buffer A (solution can be stored at room temperature for a long time): CuSO4·5H2O, 0.5 g; Na3C6H5O7·2H2O, 1 g; add double distilled water to 100 mL.
2. Buffer B (solution can be stored at room temperature for a long time): Na2CO3, 20g; NaOH, 4g; add double distilled water to 1L.
3. Buffer C: Buffer A, 1 ml; Buffer B, 50 mL.
4. Buffer D: Folin-phenol reagent, 10 mL; double distilled water, 10 mL.
Operation method:
1. Using distilled water as a blank control, the standard protein BSA was diluted to 500 ug/mL, 250 ug/mL, 125 ug/mL, 62.5 ug/mL, 31.25 ug/mL, and the samples to be tested were appropriately diluted, 40 uL per tube. The selection of the standard protein has a great influence on the experimental results. Generally, it is better to select a sample similar to the target protein as the standard protein. For example, when the antibody concentration is determined, the result of selecting the same animal IgG as a positive control is more accurate than the BSA result.
2. Add buffer C 200uL, mix and react at room temperature for 5~10 minutes.
3. Add buffer D 20uL, mix and react at room temperature for 20~30 minutes.
4. Determine the A750 value. Make a standard curve and calculate the concentration of the protein to be tested based on the A750 value of the sample to be tested.
Third, the ultraviolet absorption method
In the protein molecule, the benzene ring of tyrosine, phenylalanine and tryptophan residues contains a conjugated double bond, which makes the protein have ultraviolet absorbing properties, and has maximum light absorption at 280 nm, and its absorbance and protein content are Just proportional. The content of these three amino acids of various proteins does not differ much, so the absorbance value of the protein solution at 280 nm can be measured to reflect the concentration of the protein. The UV absorption method is simple and fast, but the accuracy is poor. For nucleic acid-containing protein solutions, A260 should also be determined.
Operation method:
1. Prepare a blank control and a protein solution to be tested.
2. The instrument wavelength was adjusted to 280 nm.
3. The absorbance A280 was adjusted to zero with a blank control. Standard quartz Cuvettes with a diameter of 1 cm are usually used.
4. Read A280 of the protein solution to be tested.
5. Adjust the instrument wavelength to 260 nm, adjust the absorbance A260 to zero with a blank control, and read the A260 of the protein solution to be tested.
6. Calculated protein concentration: protein concentration (mg/mL) = 1.45 x A280-0.74 x A260.
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