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

Determination of DNA and Protein Content by Ultraviolet Spectrophotometry

The determination of DNA and protein content using ultraviolet spectrophotometry is a widely used analytical technique in biochemistry and molecular biology. This method relies on the principle that certain molecules, such as nucleic acids and proteins, absorb ultraviolet light at specific wavelengths. In this experiment, we focus on measuring protein concentration using UV-1700 UV-visible spectrophotometer. **Objective:** - To understand the basic principles of ultraviolet spectrophotometry for protein analysis. - To master the experimental procedures for determining protein concentration using UV spectrophotometry. - To learn how to operate the UV-1700 spectrophotometer and recognize its main components. **Principle:** UV-visible spectrophotometry measures the absorption of light by molecules in a solution. When light passes through a sample, some of it is absorbed depending on the molecular structure. Proteins contain aromatic amino acids like tyrosine and tryptophan, which have conjugated double bonds that strongly absorb light around 280 nm. This makes 280 nm an ideal wavelength for protein quantification. According to the Beer-Lambert Law (A = εbc), the absorbance (A) is directly proportional to the concentration (c) of the substance when the path length (b) and molar absorptivity (ε) are constant. By measuring the absorbance at 280 nm, we can determine the concentration of the protein in the sample. **Instrumentation and Reagents:** - UV-1700 UV-visible spectrophotometer - Cuvettes (1 cm path length) - Pipettes - Standard protein solution (5.00 mg/mL) - 0.9% NaCl solution - Unknown protein sample **Experimental Procedure:** 1. Turn on the spectrophotometer and computer, then initialize the system. 2. Select "Photometric Measurement" and set the wavelength to 280 nm. 3. Load the blank solution (0.9% NaCl) into the cuvette and perform baseline correction. 4. Prepare a series of standard protein solutions with concentrations of 0.25, 0.50, 0.75, 1.00, and 1.25 mg/mL. 5. Measure the absorbance of each standard solution at 280 nm. 6. Plot a standard curve using concentration vs. absorbance. 7. Measure the absorbance of the unknown protein sample at 280 nm and calculate its concentration based on the standard curve. **Data Analysis:** - The maximum absorption wavelength was found to be 278 nm from the absorption spectrum. - A linear regression equation was obtained: A = 0.6208C + 0.0186 with R² = 0.9998. - The average absorbance of the unknown sample was 0.674, resulting in a calculated concentration of 1.056 mg/mL. - After dilution, the final concentration of the protein solution was determined to be 3.520 mg/mL. **Conclusion:** UV spectrophotometry provides a fast, accurate, and non-destructive method for determining protein concentration. It is widely used in laboratories due to its simplicity, sensitivity, and high selectivity. This experiment successfully demonstrated the application of UV-1700 spectrophotometer in protein quantification.