UV-1200 UV spectrophotometric determination of DNA protein content - Database & Sql Blog Articles

Determination of DNA protein content by ultraviolet spectrophotometry

Ultraviolet spectrophotometric determination of DNA protein content. Analytical Instruments Co., Ltd. I. Experimental purpose  Learning the principle of ultraviolet spectrophotometry for determination of protein content;  Mastering the experimental technique for determining protein content by UV spectrophotometry;  Mastering UV-1700 UV-visible spectroscopy How to use the photometer and understand the main structure of the instrument. Second, the experimental principle UV-visible absorption spectroscopy, also known as UV-visible spectrophotometry, is to study the absorption spectrum of the molecular absorption range of 190nm ~ 750nm, based on the selective absorption of light by the material molecules in the solution. A type of analytical method. The UV-visible absorption spectrum is produced as a result of the valence electron transition of the outer layer of the molecule, and its absorption spectrum is molecular spectrum. Qualitative analysis: qualitative analysis using UV-visible absorption spectroscopy generally uses spectral comparison. The absorption spectrum characteristics of the unknown pure compound, such as the number, position, relative intensity, and shape of the absorption peak, are compared with the absorption spectra of known pure compounds. Quantitative analysis: The basis of quantitative analysis by UV-visible absorption spectroscopy is Lambert-Beer law: A=lgI0/I=εbc, when the incident light wavelength λ and the optical path b are constant, the absorbance of the colored matter is within a certain concentration range. A is proportional to the concentration c of the substance, that is, the absorbance of the substance at a certain wavelength is linear with its concentration. Therefore, by measuring the absorbance of the solution to a certain wavelength of incident light, the concentration and content of the substance in the solution can be determined. Since the molar absorption coefficient at the maximum absorption wavelength λmax is the largest, the absorbance of λmax is usually measured to obtain the maximum sensitivity. For photometric analysis, the blank solution and the solution to be tested are respectively loaded into two absorption cells of thickness b, so that a bundle of parallel monochromatic light of a certain wavelength is not irradiated with the blank and the solution to be tested, so as to pass through the blank solution. The intensity is I0, and the light transmission intensity of the solution to be tested is I. According to the above formula, the logarithm of the ratio of I0 to I, that is, the absorbance, is directly given by the instrument. Ultraviolet-visible spectrophotometer: The instrument used in UV-visible absorption spectroscopy is called spectrophotometer. Its main components are composed of five parts, namely light source, monochromator, absorption cell, detector, signal display; The composite light is split by a monochromator to obtain parallel monochromatic light of any desired wavelength. The monochromatic light is absorbed by the sample sample in the sample cell and then passed through a photodetector such as a photocell or a photomultiplier tube. The photocurrent, the generated photocurrent, is directly read out from the signal display by the absorbance A. The visible light region uses a tungsten light source and a glass absorption cell; the ultraviolet light region uses a xenon light source and a quartz absorption cell. In this experiment, the protein content was determined by ultraviolet spectrophotometry. The benzene ring of the tyrosine and tryptophan residues in the protein contains a conjugated double bond. Therefore, the protein has the property of absorbing ultraviolet light, and its maximum absorption peak is located near 280 nm (different protein absorption wavelengths are slightly different). At the maximum absorption wavelength, the relationship between the absorbance and the concentration of the protein solution is subject to Lambert-Beer's law. The assay has the advantages of simple sensitivity, rapid and high selectivity, good stability, easy to eliminate interference without consuming samples, and low concentration of salts without interfering with the measurement. Third, the instrument and reagent UV-1700 US instrument - UV-visible spectrophotometer, colorimetric tube, pipette standard protein solution: 5.00mg.mL-1 solution 0.9% NaCl solution, protein solution to be tested IV, experimental steps 1. Preparation 1. Start the computer, turn on the power switch of the main unit, start the workstation and initialize the instrument. 2. Select the measurement item (spectral scan, photometric measurement) on the working interface. This experiment selects the photometric measurement and sets the measurement conditions (measurement wavelength, etc.). 3. Place the blank in the measurement cell, click START to scan the blank, and click ZERO to zero. 4. Production of standard curve. <2> Measurement work 1. Draw the absorption curve: Pipette 2 mL of 5.00 mg/mL standard protein solution into a 10 mL colorimetric tube with a pipette, dilute to the mark with 0.9% NaCl solution, and shake well. The absorbance was measured in a range of 190 nm to 400 nm using a 1 cm quartz cuvette with a 0.9% NaCl solution as a reference. 2. Preparation of standard curve: Pipette 0.5, 1.0, 1.5, 2.0, 2.5mL 5.00mg.mL-1 standard protein solution into 5 10mL colorimetric tubes, and dilute to the mark with 0.9% NaCl solution. Shake well. The absorbance of each standard solution was measured at 280 nm using a 1 cm quartz cuvette with a 0.9% NaCl solution as a reference. 3. Sample measurement: Take an appropriate concentration of 3 mL of the protein solution to be tested, and measure the absorbance at 278 nm according to the above method. Three replicates were taken in parallel. V. Data processing: 1. With the wavelength as the abscissa and the absorbance as the ordinate, draw the absorption curve and find the maximum absorption wavelength. From the absorption curve, the maximum absorption wavelength λmax=278nm (not shown) can be obtained. 2. The standard protein solution concentration is plotted on the abscissa and the absorbance is plotted on the ordinate. Standard solution concentration C (mg/mL) absorbance A0.250.1710.500.3350.750.4821.000.6371.25 standard solution concentration C (mg/mL) y=0.6208x+0.0186R2=0.9998 concentration C-absorbance A standard curve equation is: A =0.6208*C+0.01863. The concentration of the protein to be tested is determined from the standard curve based on the absorbance of the sample protein solution. Parallel measurement times Sample liquid absorbance A Sample solution concentration C (mg/mL) 10.6761.05920.6721.05330.6741.056 Average concentration of the solution measured: C=(C1+C2+C3)/3=1.056mg/mL Standard deviation of measurement: S=√Σ(Xi-X)2/(n-1)=0.003 The concentration of the protein solution to be tested is: 1.056 mg/mL*10 mL/3 mL=3.520 mg/mL.