Pulse-chase analysis (PCA) is used to study the life cycles of proteins. Pulse-chase analysis experiments use radioactive and cytotoxic labels to "tag" proteins. Commonly used methods include treating cells with cycloheximide (CHX) to stop protein synthesis or radioisotopic amino acids or proteins such as green fluorescent protein (GFP). These labels are used to study proteins through their life cycles. While pulse-chase analysis is mainly used to study proteins, it can also be used to study different molecular structures that interact with proteins. Proteins can interact with different structures either because they are incorporated into the structure, such as in cells, or because they are part of a larger structure, such as in macromolecules. In biochemistry and molecular biology, a pulse-chase analysis is a method for examining a cellular process occurring over time by successively exposing the cells to a labeled compound (pulse) and then to the same compound in an unlabeled form (chase).
Mechanism Pulse-chase experiments are divided into two parts- a "pulse" and a "chase." In the "pulse" part of the experiment, the proteome of cells are labelled with radioactive amino acids. In the "chase" part of the experiment, cells are stopped from taking up amino acids. To start a pulse-chase experiment, cells are grown in the presence of radioactive amino acids. This is done so that cells will uptake the amino acids into their proteomes. When researchers want to study protein synthesis, (e.g. folding, transport, degradation), researchers start the "chase" part of the experiment. To initiate chase, cells are exposed to nonradioactive amino acid isotopes to halt the uptake of radioactive isotopes. Researchers then study the time of the chase while the protein is in the process of interest. A selected cell or a group of cells is first exposed to a labeled compound (the pulse) that is to be incorporated into a molecule or system that is studied (also see pulse labeling). The compound then goes through the metabolic pathways and is used in the synthesis of the product studied. For example, a radioactively labeled form of leucine (3H-leucine) can be supplied to a group of pancreatic beta cells, which then uses this amino acid in insulin synthesis. Various other experimental techniques can be used to supplement pulse-chase analysis. These include cell staining, immunoprecipitation, and SDS-PAGE. Shortly after introduction of the labeled compound (usually about 5 minutes, but the actual time needed is dependent on the object studied), excess of the same, but unlabeled, substance (the chase) is introduced into the environment. Following the previous example, the production of insulin would continue, but it would no longer contain the radioactive leucine introduced in the pulse phase and would not be visible using radioactive detection methods. However, the movement of the labeled insulin produced during the pulse period could still be tracked within the cell.
Removal of radioactive and cytotoxic materials PCA uses radioactive materials to label proteins in the "pulse" part of the experiment and cytotoxic materials in the "chase" to stop protein synthesis. This is hazardous to the cells that are used during experiments. Researchers have developed various methods to use materials that are not toxic or radioactive. Examples of this include using L-azidohomoalanine (AHA) and 4sU. In the example of AHA, AHA is used to label proteins. AHA then reacts with an alkyne group to isolate AHA-labelled proteins. In this method, mammalian cells are washed with 0.5%-SDS RIPA buffer and PBS, and half-life is calculated with half-life and exponential decay formulas. Protein misfolding and heat shock were induced in cells, and cells were then subject to pulse-chase analysis, SDS-PAGE, and immunoblotting to determine protein behavior. AHA is shown to be a suitable alternative to radioactive and cytotoxic materials. It has comparable results to radioactive pulse-chase analysis; the only difference detected was when using mammalian cells, as AHA was shown to possibly alter heat shock response. Cells can be studied further by studying the proteins used, post-translational modifications, and heat shock to determine cell viability. Pulse-chase analysis is also used with 4sU. miRNAs are used in post-transcriptional gene regulation, and play a large role in the cell cycle. Although miRNA is a large part of post-translational modifications, not much is known about how it degrades. When comparing initial amount of miRNA in a PCA versus at the end of the experiment, there is a significant decrease in the amount of miRNA. Although miRNA is structurally stable, indications of degradation and decay are found through determining the half-life of miRNA. In this PCA, miRNA was tagged with 4sU and were separated based on their "age" or whether they were pri-miRNAs or mature miRNAs. With 4sU being the label, mature miRNAs were separated from pri-mRNAs after protein processing based on the amount of degradation on labels. The efficiency of miRNAs can also be determined through pulse-chase analysis by comparing transcription rates between pri-miRNA and mature mRNA.
Impact of material on decay rates In PCA experiments, proteins kinetics are interpreted by studying the length of a chase. While proteins degradation often follows exponential models of decay, problems in predicting decay curves occur when degradation does not follow an exponential model. Proteins can degrade over time without external factors, but cytotoxic and radioactive materials used in pulse-chase experiments increase the rate of degradation. Decay patterns are determined from the amount of degraded protein at the end of a chase. For this reason, accurate degradation is important to determining decay rate. To account for non-exponential decay patterns, pulse length and probabilities of molecular decay are taken into consideration. After experimental data is collected, decay rates are shown using Markov chains. Markov chains are statistical methods to determine the probability of an event. In PCA, Markov chains are used to predict the lifetime of a molecule, the age-dependent decay rate, and accurate pulse length.
Methods used with PCA
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