In this experiment, a SDS-PAGE gel was used to analyze the protein samples from the MBP-AP and WT-AP experiments. The samples are then referenced to the ladder to determine the molecular weight of the MBP-AP and WT-AP proteins. Then the UV absorbance of the two proteins from 240 nm to 340 nm is determined using a nanovolume cuvette. The absorbance at 280 nm was then used in conjunction with data from previous experiments to determine the concentration of the MBP-AP and WT-AP protein samples. Results of experiments showed that the SDS-PAGE gel yielded expect bands and the approximate molecular weight of wild type alkaline phosphatase and maltose binding protein-alkaline phosphatase is 49 kDa and 95 kDa, respectively. Additionally, the concentrations of MBP-AP Elution 1 protein and WT-AP Stage 4 are 0.278 mg/mL and 0.582 mg/mL, respectively.
Introduction To determine the purity of a protein, a sodium dodecyl sulfate polyacrylamide gel …show more content…
Then centrifugation is used to remove all the non-protein macromolecules, resulting in a large number of bands in the CCE lane. Most of the bands in the lane are very faint because they are not concentrated in the whole protein sample. In the Flow-Through stage, MBP-tagged AP binds to the amylose resin and the rest is eluted off the column, as reflected in the FT lane. In this stage, most of the undesired protein is removed. For Wash 1, Wash 2, and Wash 3, every last bit of undesired protein is eluted of the column. However, in Wash 2, there is a small amount of the MBP-AP protein that was eluted off the column, which mean be caused by human errors such as cross contamination. Finally, in Elution 1 and Elution 2, elution buffer was used to elute MBP-AP off the column, resulting in a strong band in E1 and E2 lanes. In addition, there are no visible impurities, which means that the protein is very
Introduction To determine the purity of a protein, a sodium dodecyl sulfate polyacrylamide gel …show more content…
Then centrifugation is used to remove all the non-protein macromolecules, resulting in a large number of bands in the CCE lane. Most of the bands in the lane are very faint because they are not concentrated in the whole protein sample. In the Flow-Through stage, MBP-tagged AP binds to the amylose resin and the rest is eluted off the column, as reflected in the FT lane. In this stage, most of the undesired protein is removed. For Wash 1, Wash 2, and Wash 3, every last bit of undesired protein is eluted of the column. However, in Wash 2, there is a small amount of the MBP-AP protein that was eluted off the column, which mean be caused by human errors such as cross contamination. Finally, in Elution 1 and Elution 2, elution buffer was used to elute MBP-AP off the column, resulting in a strong band in E1 and E2 lanes. In addition, there are no visible impurities, which means that the protein is very