Pulse-chase analysis
Encyclopedia
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). Radioactivity is a commonly used label.
). 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 B cells
, which then uses this amino acid in insulin
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 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.
kinase
C, ubiquitin
, and many other proteins. The method was also used to prove the existence and function of Okazaki fragments. George Palade used pulse-chase of radioactive amino acids to elicit the secretory pathway
.
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 labelingPulse labelling
Pulse labelling is a biochemistry technique of identifying the target molecule presence by inclusion of a pulse of a radioactive compound. This is mainly done to identify the stage at which the messenger RNA is being produced in a cell. In this the pulse of 3H atom is pulsed for a period of around...
). 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
Leucine is a branched-chain α-amino acid with the chemical formula HO2CCHCH2CH2. Leucine is classified as a hydrophobic amino acid due to its aliphatic isobutyl side chain. It is encoded by six codons and is a major component of the subunits in ferritin, astacin and other 'buffer' proteins...
(3H-leucine) can be supplied to a group of pancreatic B cells
Beta cell
Beta cells are a type of cell in the pancreas located in the so-called islets of Langerhans. They make up 65-80% of the cells in the islets.-Function:...
, which then uses this amino acid in insulin
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
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 insulin
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
would continue, but it would no longer contain the radioactive leucine
Leucine
Leucine is a branched-chain α-amino acid with the chemical formula HO2CCHCH2CH2. Leucine is classified as a hydrophobic amino acid due to its aliphatic isobutyl side chain. It is encoded by six codons and is a major component of the subunits in ferritin, astacin and other 'buffer' proteins...
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 proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
kinase
Kinase
In chemistry and biochemistry, a kinase is a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation. Kinases are part of the larger family of phosphotransferases...
C, ubiquitin
Ubiquitin
Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Among other functions, it directs protein recycling.Ubiquitin can be attached to proteins and label them for destruction...
, and many other proteins. The method was also used to prove the existence and function of Okazaki fragments. George Palade used pulse-chase of radioactive amino acids to elicit the secretory pathway
Secretory pathway
The secretory pathway is a series of steps a cell uses to move proteins out of the cell; a process known as secretion. The path of a protein destined for secretion has its origins in the rough endoplasmic reticulum, a membrane-bound compartment in the cell...
.