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Unformatted text preview: both hydrophobic and ionic interactions stabilize the anionic form of the dye,
causing a visible color change. Within the linear range of the assay (~5-25 μg/mL), the more
protein present, the more Coomassie binds. With the addition of Bradford reagent, which in
particular includes Coomassie Brilliant Blue dye, the binding of the protein causes a shift in
wavelength of maximum absorption of the dye from 465 nm to 595 nm. At this new wavelength,
then, the data points for the lower concentrations seemed to fit a linear relationship. After
throwing out an outlier data point for the highest concentration sample, the best fit line was
found to follow the equation Y = 0.0004385x + 0.0125865, where y is absorbance and x is protein concentration. The R2 value of .9911 for the line indicated relatively little deviation of the
data points from the best fit line.
The concentrations of each of the unknowns, then, were determined using this best fit
line. By finding the corresponding concentration to each of the measured absorbencies,
respectively, the actual concentration of the original unknown sample could be found by
multiplying the concentrations of the samples by their dilution factor. The average concentration
of the original unknown sample was determined to be 8038 μg/mL by averaging the four values
found for the four different samples using the methodology described above, and the standard
deviation was found to be 8038 μg/mL ± 1080 μg/mL....
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- Spring '13