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Welcome to the Novus Visual Protocol Series. In this video we will learn how to perform all phases of a Western Blot using the most common methods for this assay. Before we can start preparing the blot we must first prepare our sample lysate. In this example we will prepare a protein lysate from cultured cells. Here we wash the cells twice with ice cold PBS and enough lysis buffer to cover the cells. The choice of lysis buffer depends largely upon the localization of your protein of interest. We scrape the cells and transfer the cell solution on a centrifuge tube placed on ice. In order to solubilize membrane bound proteins, we will require stronger extraction detergents compared to isolated cytoplasmic proteins. In this example we are using a standard RIPA buffer, which is a common buffer for obtaining maximum protein yield. While extracting proteins from all cellular localizations, it is very important to include protease inhibitors in your lysis buffer which will prevent degradation
