Protein ultraviolet spectrometry assay
Protein ultraviolet spectrometry assay
Since the benzene rings of tyrosine and tryptophan residues in protein molecules contain conjugated double bonds, proteins have the property of absorbing ultraviolet light, with the peak absorption at 280 nm. In this wavelength range, the light absorption value (A280) of the protein solution is proportional to its content and can be used for quantitative determination.
Due to the nucleic acids at 280 wavelengths also have light absorption, the determination of protein has an interfering effect, but the maximum absorption peak of nucleic acids at 260nm, such as the simultaneous determination of light absorption at 260nm, through the calculation of the possible elimination of its effect on the determination of proteins, so the presence of nucleic acids in solution must be measured simultaneously with the 280nm and the optical density of the 260nm, in order to measure the concentration of proteins in the solution through the calculation. Therefore, when nucleic acids are present in the solution, the optical density at 280nm and 260nm must be measured at the same time to calculate the protein concentration in the solution.
Operation method
Protein ultraviolet spectrometry assay
Principle
Since the benzene rings of tyrosine and tryptophan residues in protein molecules contain conjugated double bonds, proteins have the property of absorbing ultraviolet light, with the peak absorption at 280 nm. In this wavelength range, the light absorption value (A280) of the protein solution is proportional to its content and can be used for quantitative determination. Due to the nucleic acids at 280 wavelengths also have light absorption, the determination of protein has an interfering effect, but the maximum absorption peak of nucleic acids at 260nm, such as the simultaneous determination of light absorption at 260nm, through the calculation of the possible elimination of its effect on the determination of proteins, so the presence of nucleic acids in solution must be measured simultaneously with the 280nm and the optical density of the 260nm, in order to measure the concentration of proteins in the solution through the calculation. Therefore, when nucleic acids are present in the solution, the optical density of 280nm and 260nm must be measured at the same time. The advantages of using ultraviolet absorption to determine protein content are rapid, simple, no sample consumption, and low concentrations of salts do not interfere with the measurement. Therefore, it is widely used in the biochemical preparation of proteins and enzymes (especially in column chromatographic separation). The disadvantages of this method are (1) there is a certain error in the determination of those proteins that differ greatly from the tyrosine and tryptophan content of standard proteins, and (2) if the sample contains UV-absorbing substances, such as purines and pyrimidines, there will be a greater interference. The UV absorption of different proteins and nucleic acids is different, and even after correction, the measurement results still have a certain error. However, it can be used as a basis for preliminary quantification.
Materials and Instruments
Standard protein solution Protein solution to be measured Move I. Reagents For more product details, please visit Aladdin Scientific website.
1. Standard protein solution
Accurately weigh the standard protein corrected by micro Kjeldahl method and prepare a solution with a concentration of 1mg/ml.
2. Protein solution to be measured
Prepare a solution with a concentration of about 1mg/ml.
II. Operation
1. Drawing of standard curve
Add all kinds of reagents to no test tube according to the table below and shake well. Choose the quartz colorimeter with an optical range of 1cm and measure the A280 value of each tube at 280nm. The A280 value was taken as the vertical coordinate and the protein concentration as the horizontal coordinate to draw the standard curve. 
2. Sample Determination
Take 1ml of the protein solution to be tested, add 3ml of distilled water, shake well, measure the light absorption value at 280nm according to the above method, and check the concentration of the diluted protein to be tested from the standard curve.
Other methods
1. Dilute the protein solution appropriately, and measure the A value at the wavelength of 260nm and 280nm, and then use the absorption difference at 280nm and 260nm to find out the protein concentration.
Calculation
Protein concentration (mg/ml) = 1.45A280-0.74A260
Where A280 and A260 are the light absorption values of the solution measured at 280 nm and 260 nm, respectively.
In addition, can also be calculated first A280 / A260 ratio, from the following table to find out the correction factor F value, at the same time can be found in the sample mixed nucleic acid percentage content, the F value will be substituted, and then by the following empirical formula for direct calculation of the solution of protein concentration.
Protein concentration (mg/ml) = F x 1/d x A280 x N
Where A280 is the value of light absorbance of the solution measured at 280nm, d is the thickness of the quartz colorimetric cup (cm), and N is the dilution of the solution.
Correction factor for protein content determination by ultraviolet absorption method 
2. For dilute protein solution, the absorption difference between 215nm and 255nm can also be used to determine the concentration. The concentration can be determined from the standard curve of absorption difference △A and protein content.
Absorption difference △ A = A215 - A225
Where A215 and A225 type protein solution in the 215nm and 225nm wavelength measured light absorption value.
This method obeys Beer's law in the range of protein content up to 20-100 μg/ml. Sodium chloride, ammonium sulfate, and buffers such as 1 × 10-1mol/l phosphoric acid, boric acid, and trimethylolaminomethane did not interfere significantly. However, buffers such as 1×10-1mol/l acetic acid, succinic acid, phthalic acid, and barbiturates have a large absorption at 215nm and cannot be applied, and must be reduced to 5×10-3mol/l to have no significant effect. As the protein ultraviolet absorption peak is often due to changes in pH, so the application of ultraviolet absorption method should pay attention to the pH of the solution, preferably with the standard curve determination of the same pH.
3. If a protein is known in the 280nm wavelength absorption value [A1%1cm], then take the protein solution at 280nm to determine the light absorption value, you can directly find the protein concentration.