Chromatography Lab

Topics: Green fluorescent protein, Protein, Bacteria Pages: 2 (615 words) Published: May 30, 2013
Chromatography Lab
1.The purpose of this lab was to observe the process of purifying the green fluorescent protein (GFP) in bacteria through hydrophobic interaction chromatography (HIC). Since GFP is very hydrophobic when the supernatant containing GFP is being purified through HIC column that contains a highly salty binding buffer the hydrophobic GFP sticks to the HIC beads. The other hydrophilic proteins pass through the column. The first day of lab we performed lysis of the bacterial cells by adding the enzyme lysozyme to our bacterial pellet and then freezing the solution. The next day of lab we thawed out our tube with our bacterial solution and set up chromatography column for HIC process by running equilibration buffer through the column. After centrifuging our microfuge tube containing the bacterial solution for ten minutes we transferred the supernatant and added the binding buffer. After the column was drained of buffer and fully prepped we placed it over another microfuge tube and ran the solution of the supernatant and the binding buffer through the column. Then we placed the column over a second tube and ran wash buffer through it. Finally we placed the column over the third tube and ran elution buffer though it. The first and second tubes we observed were clear and the third one glowed green. The third tube glowed because the elution buffer acts to decrease the salt concentration causing the hydrophobic proteins, which cause the fluorescent glow, to not stick to the beads as strongly and drip through isolating GFP’s.

2.The basic principle of HIC is that proteins have hydrophobic areas that bind to the beads in HIC columns containing high salt concentrated binding buffers. Essentially HIC purifies proteins based on their hydrophobicity. The salt in the binding buffer solution in the HIC column reduces solvation of the GFP which is what causes the hydrophobic regions to stick to the beads in the column. When the salt concentration is decreased...
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