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Molecular Fluorescence Analysis of Proteins - Lab Report Example

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Molecular absorption spectroscopy is a chemical technique that enables the qualitative and quantitative analysis of atoms or molecules by utilizing the absorption, emission and dispersion of electromagnetic radiations (Valeur & Berberan-Santos 10). The associations between…
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Molecular Fluorescence Analysis of Proteins
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As the electron returns from the lowest triplet state to the singlet ground state, a photon is released. The high sensitivity and specificity of fluorescence techniques facilitates the study of individual protein molecules. Therefore, this experiment sought to determine the source of fluorescence in serum albumin by first establishing the fluorescence of two key amino acids that make up serum albumin. Such a study would enable a detailed study of serum albumin as a protein. The experiment also aimed at establishing the concentration of riboflavin using the absorption of known concentrations of riboflavin.

Solutions of 10 µM tyrosine and tryptophan were made to volumes of 100µL each by first preparing 100 mL of 1mM solutions and diluting them to 10 µM. The applications software of the fluorescent instrument was loaded after which the condition parameters were set. An emission scan range of between 290 and 380 nm was set alongside slits of 5nm. The emission wavelength was set to 280 nm, and the emission tab was clicked to obtain the emission spectrum of the buffer solution. The spectrum was saved after which the same procedure was repeated for tryptophan.

Thereafter, an excitation spectrum for tryptophan was prepared by choosing the optimal emission wavelength according to the results of the emission spectrum. A tyrosine spectrum was obtained using similar conditions as those of the buffer. The tyrosine band was then obtained by subtracting the buffer spectrum from the tyrosine spectrum. All these steps were repeated for serum albumin making sure to save all the spectra. 1.0 mL of 10 ppm riboflavin solution was measured into a 100 mL volumetric flask and diluted using the buffer to make a 100 ppb stock solution.

Standard solutions were then prepared using clean volumetric pipettes rinsed with HPLC water. The final concentrations of the standard solutions were

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