User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/26
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Purification of Proteins
We needed to run 25mL of Buffer through the column at a pressure of .14/.15 ( the pressure tells if there is anything clogging the system) The conductivity meter reads how much salt is in the system Imidizole also contains the functional group of histidine. We needed to first run the binding buffer, followed by the elution buffer, and then the binding buffer once more through the column otherwise the column will have too much imidazole. The column only run at 5mL/minute. The nickel column of the instrument can hold 5mL and contains polymer beads. The ADA protein contains specifically a histag on the protein of 6 histidines (which is the amount necessary in order to have a night enough affinity to nickel). Histidine has an affinity to nickel and therefore will bind with it. We then put the protein on. We ran sample 1 first. The protein histidines (in the ADA) bind with the nickel in the column. To get the protein out, we use the elution buffer because the imidazole histidines compete with the histidine in the column and binds to the nickel. This results in the protein being pushed out. The purified protein was then collected in different Fracs (test tubes each containing 5mL) For sample 1, most of the protein was in Frac #2 ( as was indicated by the detector on the FPLC) and for the other full sample, most of the protein was collected in frac #8. Regardless, all the fracs were collected, labelled and stored in the refrigerator.