User:Elaine Marie Robbins/Notebook/CHEM-496/2011/09/13
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To determine the concentration of an unknown sample of maltose binding protein (MBP).
The absorbance values for the water and Bradford mixture were subtracted from those of the BSA and Bradford mixture to created corrected absorbance values that reflect the absorbance of the BSA only. Those values at 595 nm were plotted against concentration on a graph, and a line of best fit was determined.
The value for the absorbance of water and Bradford was also subtracted from MBP and Bradford. The value for the corrected MBP absorbance was plugged into the equation determined from the line of best fit in the Absorbance vs. Concentration graph [absorbance=0.0487(concentration)+0.0247]. The concentration of the unknown MBP was determined to be 1.074 mg/mL. Because the protein solution used in the experiment was diluted 1000x, the actual concentration of the protein sample is 1.074 mg/mL × 1000 = 1.074 g/mL. Converted to mol/L, the concentration of the unknown protein is 2.527×10-2M.
Also, a graph showing the absorbance of MBP only against wavelength was also made.
The solutions of BSA and Bradford reagent visibly appeared to be darker the more concentrated they were.