Extraction, Purification, and Observation of the Binding Capability of Lactate Binding Protein Through Fluorescence Spectroscopy
Imperial, Joshua A.
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Before the experiment commenced, the gene for lactate binding protein, found naturally in the bacteria Thermus thermophilis, was extracted from the bacteria and inserted into a DNA plasmid of Escherichia coli. The lactate binding protein was then extracted from the E. coli, purified, and analyzed for its purity by means of SDS-PAGE analysis. The purified protein was dialyzed and its concentration was determined by UV-Vis absorbance spectroscopy. Evidence of the binding of lactate binding protein to lactate was analyzed through tryptophan fluorescence spectroscopy. The fluorescence emission intensity readings of the protein were collected when the protein was in the presence of various concentrations of lactate at room temperature. Also, fluorescence emission intensity readings of the protein were collected when it was in the presence of various concentrations of lactate and in 1.0 mM pyruvic acid at different temperatures. According to the graphs of maximum fluorescence emission intensity versus temperature, there was no evidence that the protein was exclusively binding to lactate because the fluorescence emission response of the protein was similar in the presence of both lactate and pyruvic acid. No conclusion to determine the binding capability of lactate binding protein was made.