Background

Pulse-chase technique is a method that involves culturing cells in a medium containing labeled amino acids for a short period of time known as ‘pulse’. This results in the generation of newly-synthesized polypeptides incorporating labeled amino acids. The pulse step is followed by a ‘chase’ step in which labeling medium is washed out to terminate the labeling process and is replaced by a medium with non-labeled amino acids to allow for the quantification of the initially synthesized radiolabeled proteins at any given time during this chase phase. This technique thus allows quantitative analysis of the processing of a protein of interest from synthesis to degradation in a timely fashion. Pulse-chase method can be used to analyze a variety of processes such as protein folding, co-translational modifications and intracellular transport (Jansens and Braakman 2003; Magadán, 2014). However, pulse-chase technique is most commonly used to assess the stability of a protein under different experimental conditions.

Radioactivity is a commonly used label. Labeling is usually done for proteins using radioactive S35 Methionine. The kinetics of disappearance of the radiolabeled proteins relies on how fast these proteins get degraded which can be exploited to examine the effect of different experimental conditions on protein stability. To assess the stability of a particular protein of interest, this protein is immunoprecipitated from all other immunolabeled proteins that were produced during the pulse period using a specific antibody. Immunoprecipitation will isolate a ratio of radiolabeled protein that was produced during the pulse and has not been degraded until the time of immunoprecipitation. Then radioactivity is measured to assess the relative amounts of labeled protein over a period of time.

While the focus of this protocol is the assessment of protein stability, pulse-chase method can also be used to assess the stability of mRNA. Labeling of mRNA during the pulse step can be done using 3UTP. Quantitative analysis of mRNA of interest then follows by taking aliquots of total mRNA at different time points and applying one of two methods: 1) Dot blot method in which the labeled mRNA of interest anneals to a complementary single stranded nucleic acid attached to a filter paper, other labeled mRNA are then washed out and radioactivity is measured by autoradiogram or scintillation counting, 2) Affinity purification method in which the labeled mRNA of interest anneals to a complementary anchored RNA/DNA immobilized to beads, the beads are then collected by centrifugation and radioactivity of the captured labeled mRNA is quantified.