FPLC
Goals
Run an FPLC of the protein in order to purify it.
Make AuNP solution to run AAS
Procedure
- FPLC is Fast protein liquid chromatography and is used in order to purify the protein and uses a system of pumps in order to push the various liquids around the apparatus.
- First, the system needed to be equilibrated. The superloop pushed proteins through the system and can hold 50 mL. In A1 the tube was placed in the binding buffer and for B1 the tube was placed in the elution buffer in order to equilibrate the system.
- 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.
- Out stock concentration of BSA was 15μM and the concentration of the Au Stock was .0336M and that was diluted to 2mL of .0336M Au stock in 10mL of H20. We made the the various concentrations in the same manner as we had on 2012/08/29. These samples were then placed in the oven at 80°C for four hours.
Data
This graph shows the various fracs collected for both samples. It is labelled from 1-11 on the X axis. The peaks within the 25mL range indicate where the protein was detected and which frac most of it was placed into.
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