Insertion and Isolation of
Plasmids
Determining Protein
Concentration
PROTEIN
PURIFICATION
Theory: The main reason for doing the protein purification was to isolate the GFP from the E. coli colonies. In order to do this, one colony was chosen from the plate containing the LB medium, ampicillin, and arabinose, and was placed in more medium. (This medium was a store of nutrients that the bacteria could feed off of. It provided carbohydrates and other nutrients needed for metabolism, as well as amino acids for protein synthesis.) The centrifugation that was done allowed the protein to gather in a pellet at the bottom of the microfuge tube. After resuspending the pellet in TE buffer, the lysozyme was added. The main job of the lysozyme was to break open the bacterial cell walls in order to release the GFP gene. After freezing the sample overnight, it was centrifuged again to spin down the bacterial cell walls. The remaining GFP protein was in the supernatant. The binding buffer placed in the supernatant was able to bind to the GFP.
HIC was then used in order to further separate out the GFP gene from the rest of the protein. The support column contains a resin that is able to hold onto the GFP. It is typical to use HIC after salt precipitation because of high ionic strengths. In our experiment, the sample was placed in TE buffer and 4 M ammonium sulfate (causing high ionic strengths). The salt caused the protein to unfold, exposing the hydrophobic sites. This allowed the GFP to stick to the resin in the column. After a wash buffer was placed through the column, the GFP wasn’t able to stick as well because the proteins folded up again. TE was added last, and the concentration of water was very high. This caused lots of protein folding and no hydrophobic groups were exposed. The GFP protein fell through the column along with the TE buffer.