Alkaline Phosphatase Was Performed After The Anion Exchange Chromatography

Alkaline Phosphatase was isolated using various techniques. BCIP spot test was performed after the anion exchange chromatography was done and the fractions were collected. The picture of BCIP spot test of the 20 fractions can be seen in figure 1. The intensity of each spot was different from each other. The more intense color the spot was, the more concentrate the AP was. The fractions were chosen to combine together based on the intensity, which made the stage 4 enzyme. Different volumes in each stage were recorded. The volume could be seen in figure 2. The volume of protein decreased in each stage from 45ml to 6ml. After the isolation of the AP, two assays were done. First, Bradford assay was prepared. According to figure 3, “A” row from
It was also accounted for human error that might happen during the experiment. For the BSA standard, the concentration increased as it went down from row A to G in the column 11 and 12. It made sense that the stage 1 enzyme would have blue color due to the amount of other protein that had not been separated. The well color of stage 4 enzymes were more fainted compared the stage 1 enzyme. The 96 well plates were put in spectrometer and absorbance data at 595nm were obtained. In figure 4, the corrected absorbance at 595nm (after subtracting the background value of the buffer) was plotted against the BSA standard concentration. The equation from the graph was taken to calculate the protein concentration, where corrected absorbance at 595nm was y and protein concentration was x. The protein concentration was divided by its respective dilution factor in order to account for how much the protein was put in. Figure 5 shows the protein concentration from each dilution factors for each stage enzyme that were calculated using the equation from figure 4. The protein concentration mostly decreased from stage 1 to stage 4, as expected from the
SDS-PAGE gel image could be seen in figure 7. The first column was the protein ladder with known molecular weight for each band. The stage 1 enzyme consisted of numerous bands, showing the number of protein that could be found in the mixture. Fewer bands were seen in stage 2 and 3, meaning that the isolated techniques were working since fewer protein was in the mixture. Lastly, one band could be seen in stage 4 enzyme, which was the isolated alkaline phosphatase. The band was really fainted in stage 4 enzyme since one of the group member loaded the gel too fast causing the protein to spill out. Therefore, less protein was in the well. The commercial AP band was very dark stained since the protein concentration was higher compared to the isolated protein. The retardation factor was calculated for each band in protein ladder, stage 4 enzyme, and the commercial AP. The retardation factor of each band in the protein ladder was plotted against the logarithm of known molecular weights of each protein band, as seen in figure 8. The equation from the graph was taken to calculate the molecular weight of the isolated AP and the commercial AP where y is the logarithm of molecular weight and x is the retardation factor. The calculation of the molecular weight could be seen in the result. The molecular weight of the isolated AP was found to be 57.15 kDa and the molecular weight of the commercial AP was calculated to be 50.00kDa. The