Quantification Protocol Variations

Prit Patel

2/26/2016

Quantification Protocol Variations/Explanations

MCB 253 Section L

One change that was made with the protocol was dealing with the reagent volumes. Originally the experiment was going to be conducted with only 1 mL of Bradford Reagent in each cuvette, and that would then be mixed with buffer and unknown protein sample. If only 1 mL of Bradford reagent was going to be used then the cuvettes would not be filled up to the top. In order to fill the cuvettes to the top, the volume of Bradford Reagent being used was changed to 1.2 mL. Another change that was made to the protocol was the range of data that was going to be collected. In the original protocol a wide range of protein concentrations were going to be tested (0-2000 mcL). The data being collected was adjusted to fit a linear range of 125-1000 mcL of protein concentrations.

The volume of Bradford Reagent used was changed because it would provide more accurate results in the spectrophotometer. If the cuvette is not completely filled then the spectrophotometer would give inaccurate results. Filling the cuvette completely eliminated the possibility of the empty space effecting the absorbance value of the samples. This in turn made the data for the samples very accurate. The range of protein concentrations tested was adjusted by taking out the concentrations that didn’t fit the linear curve. The extreme concentrations such as 1500 and 2000 mcL of protein concentration do not fit the linear values like the other concentrations, so they were not used. Not having these extreme values in the data allowed for the creation of an accurate best fit line. With a best fit line not being affected by the nonlinear values allowed for a more accurate determination of unknown protein concentration.