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.
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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 with the elution buffer the proteins no longer stick as strongly to the beads and drip through to be isolated. 3.
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cells for chromatography it is important to add lysozyme because it digests bacterial cell wall which allows the GFP in the cells to be released into the solution so that it can then be isolated in the HIC process. The freezing step that follows is also important because it causes the bacteria to explode and rupture completely so it just further ensures that the GFP is released. 4. Tube 1 was clear because the binding buffer with a high salt concentration mixed with supernatant caused the hydrophobic areas in the proteins to stick to the beads so that the only thing that ran through the HIC column was the hydrophilic proteins.
The second tube was clear as well because it was another step to help wash the extra hydrophilic proteins through with the addition of the wash buffer to the column. The last tube was the only tube that glowed because elution buffer was added to the column which caused the salt concentration to decrease and the hydrophobic proteins stuck to the beads in the column less strongly and dripped through. In the third tube were the isolated green fluorescent proteins. . GFP can be used in a researcher’s research by tagging certain genes of interest or genes they want to observe with the GFP. Say they are studying specific characteristics expressed by genes they can tag the genes by inserting GFP’s. So now when they transfer the tagged GFP gene into another species they can observe its affects and know specifically what that gene does and if the species can uptake it or express it.