Final answer:
An experiment to determine the sidedness of erythrocyte membrane proteins using trypsin involves isolating erythrocyte membranes, treating with trypsin, stopping the reaction, and analyzing with SDS-PAGE to identify proteins with cleaved extracellular domains.
Step-by-step explanation:
To determine which proteins have an extracellular domain in the erythrocyte membrane, one could design an experiment using trypsin. Since trypsin can cleave the hydrophilic portions of membrane proteins but cannot penetrate the hydrophobic lipid bilayer, it can be used to selectively digest extracellular domains of membrane proteins. Here’s how you might conduct such an experiment:
- Isolate erythrocyte membranes by centrifuging and washing red blood cells.
- Expose the isolated erythrocyte membranes to trypsin, allowing the enzyme to cleave extracellular domains of membrane proteins.
- Stop the reaction with a trypsin inhibitor to ensure that only the extracellular domains are digested.
- Analyze the digested and undigested portions via SDS-PAGE, which separates proteins based on size.
- Proteins with a decrease in molecular weight on the SDS-PAGE gel can be inferred to have had extracellular domains that were cleaved by trypsin.
- Confirm the identity of the proteins that had their extracellular domains digested using mass spectrometry or western blotting with specific antibodies.
Through SDS-PAGE analysis, one can determine the size of the fragments generated by trypsin digestion. Fragments of a lower molecular weight than the intact protein are indicative of proteins with extracellular domains that were accessible to trypsin and hence digested.