User:Steven Moss/Notebook/AU CHEM-570/2012/02/21

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Objective

  • Purification and concentration of GFP

Proceduree

Purification of GFP with FPLC

  • FPLC was run with a 5mL Ni column using the previously made binding and elution buffers
  • 25mL of binding buffer (5X column volume) were run over the column
  • 25-50 mL of GFP solution were then run on the column so GFP could bind to Ni
  • 25 more mL of binding buffer were run on column
  • 25 mL of elution buffer were run over column and fraction collection was started
  • Process was repeated from beginning with 25 mL of binidng buffer over column until all GFP was collected.

Concentration of GFP

  • All fractions from FPLC that contained GFP were combined and concentrated including 3 washings with the binding buffer

Data

D1CGFP.PNG

Notes

  • All fractions containing GFP appeared green so no additional testing was done
  • Started by doing 50 mL of GFP solution per column elution and moved towards doing lower volumes closer to 25 mL.