User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/26

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Purification of Proteins

  • Purification was achieved by running the collected filtrate through an affinity chromatography that is attached to a Fast Protein Liquid Chromatography (FPLC). The affinity utilized is the affinity of the His tags to nickel.
  • The initial step is to equilibriate the system by adding 50 mL of the clear, colorless binding buffer to the superloop using a syringe. Flowpath was at the generic position Load.
  • The end of pump A was inserted to the bottle solution of binding buffer; pump B was inserted into the bottle of the elution buffer. The pump was set at pump wash basic.
  • In consecutive order, the column was flushed with 25 mL of the binding buffer, 25 mL of elution buffer, and another 25 mL of binding buffer.
  • The flowpath was switched to position 3 after the run through of buffers. The pump flow was at 0.15 MPa and the flowrate was set to 5 mL/min.

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.