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Problem 2.3.6 (Covered in Tutorial) (tags: #5DS-PAGE. #protein topology) SDS-PAGE is often used to determine the location and orientation of membrane associated proteins Commonly, experimental preparations of membranes are treated in a variety of ways in order to extract the proteins, which are then subjected to SDS-PAGE. These treatments will affect membrane proteins in different ways, based on how they are arranged in the membrane. One of the most common pre-treatments is by the digestive enzyme trypsin, which degroades polypeptide chains. Trypsin is too large to cross a membrane, but can digest exposed parts of membrane proteins. Those parts of a membrane protein that are in contact with extemal aqueous medium containing trypsin are digested, but the enzyme cannot digest the parts of the protein that are embedded within the lipid bilayer, or the parts of the proteins on the cytoplasmic side of the membrane if the cell is intact. If detergents are used to gently permeabilize the membrane preps prior to placing them in trypsin containing medium, the enzyme can access both sides of the membrane and will digest any polypeptide chains that is exposed and accessible a) Draw a portion of a plasma membrane of a cell and label the cytoplasmic and extracellular surface. Place the following four proteins in the membrane: a) peripheral membrane protein cytoplasmic exposure), b) integral protein (transmembrane exposure), c integral protein extracellular exposure] and d) peripheral protein (extracellular exposure] b) Describe what will happen to each of your proteins when this cell is exposed to trypsin only cl Describe what will happen to each of your proteins when this cell is treated briefly with a detergent first, and then exposed to trypsin. Experimental Design and Procedure: In this experiment, the proteins were extracted from the membranes with detergent and separated by SDS- PAGE. Prior to this, the cells were subjected to the following four Treatment I: Intact cells, no trypsin treatment Treatment 2: Brief detergent treatment first, no trypsin treatment Treatment 3: Trypsin treatment of intact cells. Treatment 4: Brief detergent treatment first, followed by trypsin . Membranes contain many different types of proteins. In this experiment, five different proteins were identified using antibody labeling. Each antibody will recognize its target protein, but not other proteins. The results can be seen in the image on the right d) Use the data from the figure above to fill out the table below Protein ID Integral Cytoplasmic exposure External exposure Transmembrane Cytoplasmic exposure External exposure Peripheral BIOL200 Problem Sets Page 28 of 129

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