June 19th - In the Lab

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Worked with Jonathan today. He was going to go through a purification process. To get protein, live E. Coli is used express the given protein in an incubator. Once incubated, the bacteria is centrifuged to get a ball of bacteria. The bacteria is then placed in a container that is in Liquid nitrogen to stop any proteolytic degradation (hoping I spelled that correctly) - stoping enzymes from chewing up proteins into smaller parts - since we are trying to preserve protein. The bacteria is placed in vials and protease inhibitors and a buffer are added, again to prevent damage to protein. Lysozyme is then added to lyse the bacteria - destroys the cell membrane, so opens up the bacteria and release what's inside - to get the protein that it made. The vials are then centrifuged to separate the cell parts (solid at the bottom) and the supernatant, liquid on top (containing the proteins). The liqud will be mixed with Nickel resin, prepared by Jonathan, so it can then be separated and get the protein we are interested in. Once the protein is ready it's purity will be checked through gel electrophoresis.

Gel is composed of a stacking and separating layers. To make the gel, stacking gel goes first and allowed to polymerize and then the separating gel goes second.