Question

Explain how a pulse chase experiment can be used to determine the location and order of steps in the synthesis of a protein in the cell.

Answer 1

Matthew Meselson and Franklin Stahl in 1958 conducted their now famous pulse-chase experiment.This analysis is a commonly used technique for studying the synthesis, processing and transport of proteins.It is a method to examine a cellular proces that occur over time by successively exposing the cells to a labeled compound called pulse and then to the same compound in an unlabeled form called chase.

In this experiment the cells are grown in a medium and then exposed for a short time period to a large amount of radioactive amino acid. They are commonly used to follow the intracellular location of a proteins and the transformation of metabolites during a biochemical reaction time.

The three stages of pulse-chase experiment are:-

1. The actual pulse-chase
2. Immunoprecipitation
3. Sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In the experiment, a radioactively tagged compound is used for time-based monitoring of the cellular events. After that the proteins are harvested by cell lysis through immunoprecipitation. It is then run on SDS-PAGE for further analysis.

In order to determine the subcellular location of proteins the pulse-labeled phase were fractionated into a mitochondrial fraction and a postmitochondrial supernatant.