Lab 2 - Native UV Absorbance of Biomolecules, Protein...

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Native UV Absorbance of Biomolecules, Protein Fluorescence Spectroscopy, and Bradford Protein Concentration Assay 1
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Native UV Absorbance of Biomolecules, Protein Fluorescence Spectroscopy, and Bradford Protein Concentration Assay University of Texas at Austin Department of Chemistry and Biochemistry Introduction While performing this experiment, our objectives were to measure the concentration of proteins and nucleic acids using ultraviolet spectrophotometry. We used Beer’s Law because it states that absorbance of light is proportional to concentration. Beer’s Law has the following terms: absorbance (A), which is a measure of the intensity of light that passes through a sample at a specific wavelength. The molar extinction coefficient, ε, is how strong a chemical solution absorbs light at a specific wavelength. The path length, ℓ, is the cuvette length where the light passes through and the concentration, c , is the concentration of the absorbing species. A = ε ℓc (1) The use of DNA spectrophotometry is useful in determining DNA concentrations. Nucleic acids absorb light at 260 nm so we can easily determine the relative concentrations of DNA samples. However, some amino acids have functional groups that absorb better at 280 nm rather than 260 nm. These are the aromatic amino acids, which are tyrosine, phenylalanine, and tryptophan. Since proteins contain at least one of these amino acids, the Warburg-Christian method allows us to obtain the absorbance of proteins at 280 nm. To determine the purity of a protein preparation we divide the absorbance of a protein by its nucleic acid, A 280 /A 260 . Protein Concentration (mg/mL) = (F)(A 280 )(light path in cm)(dilution factor) (2) In this experiment, we are determining the absorbance and concentration of an adenosine solution in part 1 and a DNA solution in part 2. In part 3, we are using the Warburg-Christian method to determine the absorbance and concentration of a protein. After we found the native ultraviolet absorbance of our biomolecules, we observed the
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This note was uploaded on 09/07/2009 for the course CH N/a taught by Professor Genemcdonald during the Spring '09 term at University of Texas at Austin.

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Lab 2 - Native UV Absorbance of Biomolecules, Protein...

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