Yesterday we performed a Hydrophobic Interaction Chromatography (HIC) to separate the green fluorescent protein (GFP), produced in our bacterial cultures, from other proteins commonly found in bacteria.
A sample of bacterial culture was concentrated and then resuspended in a solution in which they were lysed. The high salt solution, containing all the proteins found in the bacteria, was then passed through a hydrophobic interaction column where molecules of GFP bound to the hydrophobic beads. The high salt solution increased the hydrophobicity of GFP by further exposing its hydrophobic amino acid residues.
A series of washes with buffers of decreasing salinity allows proteins with various levels of hydrophobicity to gradually unbind from the beads and be collected in a test tube. By switching collection tubes each time a buffer is added, different proteins can be collected. One of them was GFP and the tube in which it was collected glowed with a green color (of course).
Diagram of Hydrophobic Interaction Chromatography (HIC)
GFP molecules are represented by black triangles
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