1

1. The table 10.1 in the lab manual portrays the peak 1 and 2 from the gel filtration, the absorbance at 200nm, the molecular weight, the molar absorption coefficient, and the concentration of the protein. The absorbance for peak 1 (ovalbumin) was 1.104 and for peak 2 (cytochrome c) was 0.278 and using the Beer Lambert law it was found that ovalbumin had the higher concentration (in comparison to cytochrome c (. Furthermore, table 11.2 shows the gel filtration values of peak fraction 1 & 2, their respectful absorbance, and the concentration of protein in all found using the pierce 660nm protein assay. The way in which the concentration of protein was determined was by the process of comparing it to the standard curve produced of the 8 solutions from table 10.1. Therefore, it was determined that the average concentration from the standard curve of peak 1 and 2 was 0.157 and 0.073 and this data was significant because it gives us the amount/concentration of protein in each peak fraction; it also describes the highly accurate nature of the pierce 660nm protein assay due.
2. The table 10.1 again shows the data from the respected peak 1 (ovalbumin) and 2 (cytochrome c), the molecular weight, and the concentration- all from the ion exchange column. The results reported that ovalbumin had the higher concentration (1.156*10^-5 M) and absorbance compared to cytochrome (8.37*10^-6 M). This is similar to table 11.3 where there the results show the absorbance & concentration of peak fraction 1 and 2 done through the pierce 660nm protein assay of the ion exchange column. The average concentration for peak 2 was .0245 and 0.190 for peak 1. As a result, the pierce 660nm protein assay is more accurate because it is very sensitive as the absorption maximum changes (due to interaction with proteins in an acidic environment) and so easily identifies which sample as the higher concentration.
3. Table 10.3 depicts 10 different markers, the peak 1 and 2 from both ion exchange column and gel filtration and their respected migration distance, molecular weight, and lastly Rf value. The equation for the Rf is protein migration distance (cm)/dye front migration distance (cm). For gel filtration, peak 1(ovalbumin) had a migration distance of 3.0 and for peak 2(cytochrome c) had a migration distance of 6.3. This data is significant and makes sense since in both gel filtration and ion exchange, cytochrome c traveled further down the plate than ovalbumin due to the lower weight of cytochrome c. Additionally, the data from table 10.3 was used to make a semi-log graph of molecular weight vs Rf value and was used to find the molecular weight of cytochrome c and ovalbumin. Therefore, the molecular weight of ovalbumin was 42 Daltons and that of cytochrome c was 15 Daltons.
4. The main advantage of SDS-Page gel compared to immunoblot is that SDS-Page gel identifies all your proteins in the sample, while immunoblot is more specific to the target it identifies. SDS-Page analysis is sensitive to proteins that fall in the range of 25 to 2,000 and the advantage is that the presence of reducing agents/detergents do not interfere with the assay (Hayden-Mcneil). On the other hand, due to the use of antibodies by immunoblot it is more specific to your target protein you want to identify. Lastly, it is significant to note one similarity between SDS-Page and immunoblot is that ovalbumin, cytochrome c, and b-galactosidase all eluded in the same relative order.