Affinity Chromatography Lab Report
Figure 1: Affinity chromatography of fumarase with the Ni2+-NTA-agarose column.
Extract (9.9 mL) containing yeast (3.76g) in extraction buffer containing 0.1% Igapel CA-630 and protease inhibitors were pumped through Ni2+-NTA-agarose column. Fractions were collected by 1.5 mL portions by use of wash buffer (20.0 mL), imidazole elution buffer (26.3 mL), and wash buffer (10.0 mL), again. Absorption readings were taken for all fractions with a Cary50 set at 280nm. The fumarase activity was determined by the highest absorbances using the Cary50 set at 250nm. The pre-dialysis elution pool was pooled together from fractions 17-18 and the flow-through pool was determined to be fractions 2-9.
Figure 2: Bradford standard curve created by an assay of seven known dilutions of bovine serum albumin (BSA). The assays were done at 595nm using a …show more content…
Trials of other resins in the affinity column would determine which resin would have the greatest outcome in the purification process. The Ni2+-NTA-Agarose resin was effective for this experiment, but testing other resins can determine if the Ni2+-NTA-Agarose resin was the best resin to use in this purification process. It also needs to be determined if running the protein through the column would have any effect on the dialysis process. This information can be determined by running dialysis with the crude sample and comparing the fold purity to the post-dialysis elution pool. Another future experiment, would be to determine which type of membrane used in the dialysis could affect the outcome of the fold purity. Repeating this experiment and keeping everything the same, but using different membranes in the dialysis, would determine which membrane would have the greatest impact on the fold purity. With these results, a better look can be given of how the affinity column and the dialysis affect the purification
Extract (9.9 mL) containing yeast (3.76g) in extraction buffer containing 0.1% Igapel CA-630 and protease inhibitors were pumped through Ni2+-NTA-agarose column. Fractions were collected by 1.5 mL portions by use of wash buffer (20.0 mL), imidazole elution buffer (26.3 mL), and wash buffer (10.0 mL), again. Absorption readings were taken for all fractions with a Cary50 set at 280nm. The fumarase activity was determined by the highest absorbances using the Cary50 set at 250nm. The pre-dialysis elution pool was pooled together from fractions 17-18 and the flow-through pool was determined to be fractions 2-9.
Figure 2: Bradford standard curve created by an assay of seven known dilutions of bovine serum albumin (BSA). The assays were done at 595nm using a …show more content…
Trials of other resins in the affinity column would determine which resin would have the greatest outcome in the purification process. The Ni2+-NTA-Agarose resin was effective for this experiment, but testing other resins can determine if the Ni2+-NTA-Agarose resin was the best resin to use in this purification process. It also needs to be determined if running the protein through the column would have any effect on the dialysis process. This information can be determined by running dialysis with the crude sample and comparing the fold purity to the post-dialysis elution pool. Another future experiment, would be to determine which type of membrane used in the dialysis could affect the outcome of the fold purity. Repeating this experiment and keeping everything the same, but using different membranes in the dialysis, would determine which membrane would have the greatest impact on the fold purity. With these results, a better look can be given of how the affinity column and the dialysis affect the purification