The goal for the present experiment was to purify one of the largest populations of antibodies and soluble proteins, Immunoglobin G (IgG) from rabbit serum. In addition, IgG was purified through Binding affinity chromatography. Due to proteins A and G having a high affinity for IgG a mixture of protein A and G was added to the column as it selectively binds to IgG while a mild buffer was used to wash away unbound protein molecules.
Dilutions of bovine serum albumin (BSA) of known concentrations were produced. In addition, the sample that was obtained from the affinity chromatography where IgG was isolated was then separated into two different fractions. Additionally, Bradford-Coomassie was used as a binding agent, which was added to the known protein concentration samples and the two unknown protein concentration fractions. Furthermore, three standard curve graphs (linear trendline, logarithmic trendline, second-order polynomial trendline) were generated using the BSA concentrations in order to find the best-fit trendline. As a result, the trendline that best fit and had the strongest regression value was the second-order polynomial trendline graph (R2 = 0.94525). However, the linear trendline graph also had a very strong regression value (R2 = 0.94062) illustrating a strong relation between absorbance and protein concentration. Since both the second-order polynomial trendline graph and linear trendline graph had very close regression values and both portrayed a strong relation between the absorbance values and protein concentrations it allowed some flexibility when selecting the trendline to calculate the concentration of IgG samples. Thus, the linear trendline graph was selected due to its more simple and famili...
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...stabilization of the CH2 region of the molecule. As a result, this destabilization caused a huge change in the quaternary structure causing a conformational change (2). This is evident on the present study as two different protein stains are shown on tube 2 (Figure 1) as the quaternary structure has broken off in two heavy chains polymers and two light chains polymers.
A limitation of this experiment would be when obtaining the unknown protein concentrations it is not reliable or useful to compare it with another unknown protein concentration that was obtained some other time or on a different time. Each unknown is unique and cannot be duplicated in order to obtained the same exact amount of unknown protein concentration that was obtained at first. Thus, in order to obtain accurate results in the experiment everything must be processed and obtained on the same day.
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