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
1 to 8 consisted of the 4 stage enzymes diluted with buffer and is known as dilution factor x1. “B” row is dilution factor x2.5, and “C” row is dilution factor x5. The purpose of having different dilution factor was to take into consideration of whether the protein concentration failed into the range of the standard or not.
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
intensity of color in figure 3. However, regarding the dilution factor x1, the protein concentration increased from stage 2 to stage 3, similarly to dilution factor x5. Human error in pipetting small amount of protein might be taken into consideration. If the amount of protein in each stage enzyme was to be compared to each other from different dilution factor, according to figure 6, stage 1 and 2 were varied greatly from each other. These variations might cause by the outlier of the dilution factor x1 and dilution x5 since the protein concentration from these dilution factors were not within the range of the standard. Stage 3 in different dilution factors had small differences compared to one another, similarly to stage 4 enzyme. Ultimately, most error might be from the error caused in the BSA standard since one of the group members accidentally spilled Bradford assay from the pipette while putting into the wells. Hence, the protein concentration changed due to the amount of Bradford assay was added. Another assay was also done on the purified AP.
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
percentage difference was 12.5%. The error might be due to the light color band of the stage 4 enzyme, which made it difficult to measure the protein distance. Another reason for the error was that the eight band of the protein ladder run with the dye front. Hence, the corrected measurement of protein distance of that particular band could not be made. As a result, it influenced the trend line equation that was used to calculate the molecular weight. The alkaline phosphatase was isolated successfully from the mutant K12 E-coli bacteria. The quantity and quality of the isolated AP was determined by using two different assays: Bradford assay and SDS-PAGE, proving the amount AP was isolated and the purity of AP.