Pulse-chase analysis of auxin signal transduction in Arabidopsis thaliana wildtype and mutant

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

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 Leucine (symbol Leu or L) is an essential amino acid that is used in the biosynthesis of proteins. Leucine is an α-amino acid, meaning it contains an α- amino group (which is in the protonated −NH3+ form under biological conditions), an α- ...

(³H-leucine) can be supplied to a group of pancreatic beta cells, which then uses this amino acid in

insulin Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism ...

synthesis. 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

would continue, but it would no longer contain the radioactive

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.

Uses

This method is useful for determining the activity of certain cells over a prolonged period of time. The method has been used to study

protein kinase C In cell biology, Protein kinase C, commonly abbreviated to PKC (EC 2.7.11.13), is a family of protein kinase enzymes that are involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of serine and ...

ubiquitin Ubiquitin is a small (8.6 kDa) regulatory protein found in most tissues of eukaryotic organisms, i.e., it is found ''ubiquitously''. It was discovered in 1975 by Gideon Goldstein and further characterized throughout the late 1970s and 1980s. Fo ...

, and many other proteins. The method was also used to prove the existence and function of Okazaki fragments.

George Palade George Emil Palade (; November 19, 1912 – October 7, 2008) was a Romanian cell biologist. Described as "the most influential cell biologist ever",

used pulse-chase of radioactive amino acids to elucidate the

secretory pathway 440px Secretion is the movement of material from one point to another, such as a secreted chemical substance from a cell or gland. In contrast, excretion is the removal of certain substances or waste products from a cell or organism. The classica ...

References

Cell imaging {{molecular-biology-stub