David is attempting to develop vesicles of plasma membrane that are turned insid

Question

David is attempting to develop vesicles of plasma membrane that are turned inside-out (he wants the cytosolic surface to face the inside the vesicle, and the extracellular surface to face the outside of the vesicle). Despite his best efforts, David keeps getting preparations that are contaminated with a small number of vesicles that have the orientation that would normally be seen in the cell (which is not what he wants). David’s friend, Sushmita, suggests that he uses a variation of a technique known as affinity chromatography. To do this experiment, David will need to pass the preparation containing the vesicles through a column containing immobilized beads that are coupled to a protein which binds various carbohydrate chains. David is excited about this experiment because he thinks Sushmita’s suggestion is an excellent idea. Why do you think this technique should allow David to get a pure preparation of inverted vesicles? David is attempting to develop vesicles of plasma membrane that are turned inside-out (he wants the cytosolic surface to face the inside the vesicle, and the extracellular surface to face the outside of the vesicle). Despite his best efforts, David keeps getting preparations that are contaminated with a small number of vesicles that have the orientation that would normally be seen in the cell (which is not what he wants). David’s friend, Sushmita, suggests that he uses a variation of a technique known as affinity chromatography. To do this experiment, David will need to pass the preparation containing the vesicles through a column containing immobilized beads that are coupled to a protein which binds various carbohydrate chains. David is excited about this experiment because he thinks Sushmita’s suggestion is an excellent idea. Why do you think this technique should allow David to get a pure preparation of inverted vesicles?

Explanation / Answer

Answer:

The affinity column contains ConA copolymerized with bovine serum albumin (BSA) which reacts only with RO (rightside out), because it is on the external surface of the plasma membrane that the carbohydrate residues which react with ConA are located.

The inside out vesicles would not bind and can be collected from the flow through.

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