Trypsin is an enzyme that can digest the hydrophilic portions of membrane proteins, but it is...

Trypsin is an enzyme that can digest the hydrophilic portions of membrane proteins, but it is unable to penetrate the lipid bilayer and enter a cell. Due to these properties, trypsin has been used in conjunction with SDS-PAGE to determine what proteins have an extracellular domain. Describe an experiment using trypsin to determine the sidedness of proteins of the erythrocyte membrane. BE SURE TO INCLUDE ANY RELEVANT CONTROLS

Expert Solution

Intact erythrocyte is chosen that has proteins both on the cytoplasmic side as well as the outer portion of the membrane. These are then treated with trypsin.

Trypsin cannot enter the inner side of the cell and thus hydrolysis only those parts of the protein that are facing the outer side of the membrane. A primary antibody against the protein is used and the secondary antibody used is fluorescent-tagged for visualization. If the protein is present on the cytoplasmic face of the cell, the trypsin cannot access it and it remains intact. thus the primary and secondary antibody can bind to it and give fluorescence.

In the case of the protein being on the extracellular side, trypsin has access to it and thus breaks the protein down, so the primary and secondary antibody can no longer recognize it and give fluorescence.

thus no fluorescences indicates the protein being on the side (extracellular side)where trypsin has access whereas a fluorescence indicates the protein being on the cytosolic side of the membrane where trypsin has no access.

gladiator answered 2 years ago

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