Today we lysed our bacteria open, extracted all the molecules inside them, including all their proteins, and then purified our protein of interest, the GFP.
We performed a protein chromatography using Bio-Rad's disposable columns. Our task was separating the GFP from the thousands of other proteins that we would find in a bacterium. The resin used in these columns were chosen to separate specifically the GFP by Hydrophobic Interaction Chromatography (HIC).
In HIC the resin is very hydrophobic. The salty buffer used causes proteins to change their 3-D conformation exposing the hydrophobic amino acids, wich stick to the hydrophobic resin. The GFP sticks especially strongly to the resin. Then we did washes with solutions of decreasing salinity so other proteins that happened to be less hydrophobic than the GFP recovered their original conformations, un-bound from the resin and were washed out. At the end we used a low salinity solution that allowed thr GFP to recover its original 3-D conformation making its hydrophobic amino acids un-available and separating from the resin.
Following this protocol we purified the GFP exclusively, and that's why our little tubes glowed when exposed to UV light.
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