Western Blodding, Part 1, Protein Isolation. In this animation, we will introduce you to an experimental technique called Western Blodding. Western Blodding is a technique that allows researchers to detect changes in a particular protein of interest such as changes in concentration, addition or subtraction of small chemical modifications and changes in interactions with other proteins. In the Western Blodding procedure, proteins are first isolated from the rest of the cells components. To extract proteins from cells, researchers use chemical-based and or physical-based methods to disrupt the cells plasma membrane and release the proteins inside it. Chemical-based lysis uses various types of detergents to make proteins accessible. For example, weak detergents are used to extract water-soluble proteins inside the cell without disrupting intracellular membrane compartments. Strong detergents are able to solubleize or make available the proteins found within all membranes. Following lysis, cells in their contents are spun in a microcentrifuge. This results in an aqueous supernatant which contains soluble-ized proteins and a pellet, which contains membranes, organelles, nucleic acids, and any remaining insoluble-ized proteins. The supernatant, now called a protein lysate, is separated from the pellet to use for Western Blodding. The most common type of protein lysate that researchers prepare is called a whole cell lysate because it contains all of the proteins within a cell. Two other commonly prepared types of lysates are nuclear and cytoplasmic lysates, which contain only nuclear and cytoplasmic proteins respectively, often prepared to study the regulation of protein localization to these two compartments. A fourth commonly used lysate type is an amino precipitate or IP. An IP is performed to identify and detect proteins that may interact with a protein of interest. To perform an IP, the protein of interest is isolated from a protein lysate by the addition of an antibody that specifically recognizes the protein of interest, followed by a secondary antibody conjugated to beads used to precipitate the protein antibody complex out of the lysate solution. In this manner, the researcher isolates the protein of interest along with any other protein or proteins with which it interacts physically. Cell lysate disrupts the carefully controlled cellular environment. This can cause the unwanted addition of protein modifications, protein unfolding or degradation by allowing certain enzymes involved in these processes to have unregulated access to their targets. To prevent unwanted modifications, unfolding and degradation, lysate is performed at a low temperature and in the presence of various enzymatic inhibitors. Most in blotting can be divided into two phases. In the first phase, proteins are loaded onto a gel and separated from one another using an electrical current during polyacrylamide gel electrophoresis or page. Then, in the second phase, the separated proteins are transferred onto a membrane and the protein of interest is visualized using a protein specific antibody. In the next section of this animation, we will take a closer look at polyacrylamide gel electrophoresis or page.