User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/10/02

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FPLC

Goals

Run an FPLC of the protein in order to purify it.

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. Histidine has an affinity to nickel and therefore will bind with it.



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