In a cellfree protein synthesis system utilizing microsomes

In a cell-free protein synthesis system utilizing microsomes from fragmented ER, under which condition could you determine if the new protein was imported into the microsome?

Ribosomes and mRNA are incubated with microsomes, then a protease is added and the results are analyzed.

Ribosomes and mRNA are incubated with microsomes, then a protease and detergent are added and the results are analyzed.

Ribosomes and mRNA are incubated with protease, then microsomes are added and the results are analyzed.

Ribosomes and mRNA are incubated with microsomes, then detergent is added and the results are analyzed.

Solution

A cell free system is often used to investigate the functional role of one or more components of a cellular system. One such example of cell free system includes translation of proteins and transport thereof in microsomes. Technically speaking, microsomes are small fragmented segments of rough endoplasmic reticulum which contain ribosomes attached to their surface. In this sense, the microsomes represent not only the site of protein synthesis but owing to their small size, they also help in transport of these proteins to their target location. Thus, a simple cell-free extract comprising of ribosome, rough/smooth endoplasmic reticulum, the mRNA and microsome would be capable of synthesis of a protein and its microsome mediated transport. Microsomes also contain enzymes which help in degradation of fatty acids and toxins ingested in the cell.

Thus, according to the question, the aim is to determine if a newly synthesized protein was imported into a microsome fragmented from ER. For this analysis, it must be acknowledged that when ribosomes and mRNA would be mixed, protein synthesis will take place. This protein or polypeptide would be inserted into the microsome. Now the aim is to determine whether this microsome contains polypeptide/protein or not. For this purpose, simple SDS-PAGE can be run. In order to do this, the microsome must be first treated with a protease so that the internalized protein might be degraded into smaller fractions of amino acids/polypeptides and then a detergent is applied so that all of these amino acids/polypeptides might achieve a net negative charge. Thus, these amino acid/polypeptide sequences then can be made to run on a SDS-PAGE. After staining with coomassie brilliant blue dye, appearance of bands in this sample would determine that proteins were present in the microsomes.

Thus, this explanation states that choice b is correct.