pulse-chase analysis
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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 Cycloheximide is a naturally occurring fungicide produced by the bacterium '' Streptomyces griseus''. Cycloheximide exerts its effects by interfering with the translocation step in protein synthesis (movement of two tRNA molecules and mRNA in re ...
(CHX) to stop protein synthesis or radioisotopic
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...
s 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 A proteome is the entire set of proteins that is, or can be, expressed by a genome, cell, tissue, or organism at a certain time. It is the set of expressed proteins in a given type of cell or organism, at a given time, under defined conditions. P ...
of cells are labelled with radioactive
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...
s. 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 presence of radioactive amino acids. This is done for that cells will uptake the amino acids into their proteomes. When researchers want to study the life of the cell (e.g. folding, transport, degradation), researchers start the "chase" part of the experiment. To initiate chase, cells are put in presence of a nonradioactive isotope to stop uptake of radioactive isotope. 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 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-Car ...
(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 insulin (''INS)'' gene. It is the main Anabolism, anabolic hormone of the body. It regulates the metabol ...
synthesis. Various other experimental techniques can be used to supplement pulse-chase analysis. These include cell staining,
immunoprecipitation Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sam ...
, and
SDS-PAGE SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a Discontinuous electrophoresis, discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular m ...
. 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 (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the insulin (''INS)'' gene. It is the main Anabolism, anabolic hormone of the body. It regulates the metabol ...
would continue, but it would no longer contain the radioactive
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-Car ...
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 Radioactive decay (also known as nuclear decay, radioactivity, radioactive disintegration, or nuclear disintegration) is the process by which an unstable atomic nucleus loses energy by radiation. A material containing unstable nuclei is conside ...
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 Buffer may refer to: Science * Buffer gas, an inert or nonflammable gas * Buffer solution, a solution used to prevent changes in pH * Lysis buffer, in cell biology * Metal ion buffer * Mineral redox buffer, in geology Technology and engineeri ...
and PBS, and half-life is calculated with
half-life Half-life is a mathematical and scientific description of exponential or gradual decay. Half-life, half life or halflife may also refer to: Film * Half-Life (film), ''Half-Life'' (film), a 2008 independent film by Jennifer Phang * ''Half Life: ...
and
exponential decay A quantity is subject to exponential decay if it decreases at a rate proportional to its current value. Symbolically, this process can be expressed by the following differential equation, where is the quantity and (lambda Lambda (; uppe ...
formulas. Protein misfolding and
heat shock The heat shock response (HSR) is a cell stress response that increases the number of molecular chaperones to combat the negative effects on proteins caused by stressors such as increased temperatures, oxidative stress, and heavy metals. In a norm ...
were induced in cells, and cells were then subject to pulse-chase analysis, SDS-PAGE, and
immunoblotting The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecti ...
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 modification In molecular biology, post-translational modification (PTM) is the covalent process of changing proteins following protein biosynthesis. PTMs may involve enzymes or occur spontaneously. Proteins are created by ribosomes, which translation (biolog ...
s, and
heat shock The heat shock response (HSR) is a cell stress response that increases the number of molecular chaperones to combat the negative effects on proteins caused by stressors such as increased temperatures, oxidative stress, and heavy metals. In a norm ...
to determine cell viability. Pulse-chase analysis is also used with 4sU. miRNAs are used in post-transcriptional
gene regulation Regulation of gene expression, or gene regulation, includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA). Sophisticated programs of gene expression are wide ...
, and play a large role in the cell cycle. Although
miRNA Micro ribonucleic acid (microRNA, miRNA, μRNA) are small, single-stranded, non-coding RNA molecules containing 21–23 nucleotides. Found in plants, animals, and even some viruses, miRNAs are involved in RNA silencing and post-transcri ...
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 Micro ribonucleic acid (microRNA, miRNA, μRNA) are small, single-stranded, non-coding RNA molecules containing 21–23  nucleotides. Found in plants, animals, and even some viruses, miRNAs are involved in RNA silencing and post-trans ...
. 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 In probability theory and statistics, a Markov chain or Markov process is a stochastic process describing a sequence of possible events in which the probability of each event depends only on the state attained in the previous event. Informally, ...
. 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


Cell Staining

Cell division orientations can be determined using PCA and cell staining. Cell staining is used in conjunction with PCA to support and give visual data on hypothesized cell processes. Different parts of cells, such as the spindle, are stained in order to image them at different stages of the cell cycle. This information is used with data for corresponding "pulse" and "chase" periods. In this particular experiment, 5-ethynyl-2'-deoxyuridine (EdU) is used to label ''Arabidopsis Thaliana'' leaf cells. Cells nuclei were stained and the staining and pulse-chase signals were compared. The division angle of the cells were used to determine the cell stages the direction of cell division. The morphology of cell organelles can be studied when using PCA to determine the stage of the cell's life cycle. Cells can be stained with various fluorescent cell stains such as
DAPI DAPI (pronounced 'DAPPY', /ˈdæpiː/), or 4′,6-diamidino-2-phenylindole, is a fluorescent stain that binds strongly to adenine– thymine-rich regions in DNA. It is used extensively in fluorescence microscopy. As DAPI can pass through an ...
,
Propidium Iodide Propidium iodide (or PI) is a Fluorescence#Biochemistry and medicine, fluorescent intercalating agent that can be used to Staining (biology), stain cell (biology), cells and nucleic acids. PI binds to DNA by intercalating between the bases with li ...
, or Hoechst in order to image dead cells or cell nuclei. In this experiment, immunolabeling and fluorescent staining are used in conjunction with one another. Cells are labelled with EdU. The experiment was done on plant cells- specifically, root cells of ''
Arabidopsis Thaliana ''Arabidopsis thaliana'', the thale cress, mouse-ear cress or arabidopsis, is a small plant from the mustard family (Brassicaceae), native to Eurasia and Africa. Commonly found along the shoulders of roads and in disturbed land, it is generally ...
.'' Cells were isolated from plants, immunolabelled with EdU, and stained with fluorescent dyes. The comparison of imaging cells with DAPI and the PCA results showed that amounts of
Golgi Apparatus The Golgi apparatus (), also known as the Golgi complex, Golgi body, or simply the Golgi, is an organelle found in most eukaryotic Cell (biology), cells. Part of the endomembrane system in the cytoplasm, it protein targeting, packages proteins ...
increased during the late G1 part of the
cell cycle The cell cycle, or cell-division cycle, is the sequential series of events that take place in a cell (biology), cell that causes it to divide into two daughter cells. These events include the growth of the cell, duplication of its DNA (DNA re ...
.


Immunoprecipitation and SDS-PAGE

Immunoprecipitation can be used to determine the proteins present in a solution. This is done by breaking down the different components of a macromolecule the protein is part of. When conducting PCA, it is used to recover a previously labelled protein and determine where a protein is in the process of protein processing. The protein macromolecules are treated with endoglycosidases to break them down and run through SDS-PAGE to determine the identity of the individual proteins. This information of the unique proteins present is used to determine the mechanisms of a protein at a certain time. Immunoprecipitation is not always used, however, due to concerns of misidentifying labelled and unlabelled proteins. When conducting a PCA experiment, cells are washed of extracellular debris and materials that could interfere or create noise when isolating the protein of interest. However, when the materials in an experiment are washed, this can take upwards of 15 minutes. When trying to examine specific stages of biological processes, such as cell cycles or protein degradation, increased time used to wash cells could potentially interfere with the results of an experiment. Pulse-chase experiments can be modified in order to not require washing. Researchers have developed a way to used fluorescent proteins and
fluorescence resonance energy transfer Fluorescence is one of two kinds of photoluminescence, the emission of light by a substance that has absorbed light or other electromagnetic radiation. When exposed to ultraviolet radiation, many substances will glow (fluoresce) with colore ...
(FRET) to label proteins.
Nucleophilic In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they a ...
enzymes were labelled and instead of washing, noise-interfering compounds were removed through
elimination reaction An elimination reaction is a type of organic reaction in which two substituents are removed from a molecule in either a one- or two-step mechanism. The one-step mechanism is known as the E2 reaction, and the two-step mechanism is known as the E1 r ...
s. This approach to PCA can shed light on time-dependent protein reactions such as the speed of protein expression or movement.


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 t ...
,
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 19 ...
, and many other proteins. The method was also used to prove the existence and function of
Okazaki fragments Okazaki fragments are short sequences of DNA nucleotides (approximately 150 to 200 base pairs long in eukaryotes) which are synthesized discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DN ...
. George Palade used pulse-chase of radioactive amino acids to elucidate the
secretory pathway Secretion is the movement of material from one point to another, such as a secreted chemical substance from a cell (biology), cell or gland. In contrast, excretion is the removal of certain substances or waste products from a cell or organism. Th ...
.


Health Science

PCA is used in several ways for
biomedical research Medical research (or biomedical research), also known as health research, refers to the process of using scientific methods with the aim to produce knowledge about human diseases, the prevention and treatment of illness, and the promotion of ...
- it uses proteins to study the lifespans and of structures that interact with proteins. Examples of this include stem cells, procollagen, cell turnover, and viral inclusions.


Cell Division and Turnover

Pulse-chase analysis can be used to determine the rate of division of stem cells. Stem cells are different from regular cells in that they rarely divide. Researchers have used pulse-chase analysis in the kidney cells of mice and rats in order to study the cell cycle and cell life length of stem cells. Different pulse lengths were used to determine whether a label-retaining cell (LRC) was a stem cell or not. Cells that retained their tags for longer periods of time and were "slow-cycling" were hypothesized to be stem cells. Pulses were also used to detect DNA analog labels, and to detect the number of cell divisions they had gone through by using the cells'
half-life Half-life is a mathematical and scientific description of exponential or gradual decay. Half-life, half life or halflife may also refer to: Film * Half-Life (film), ''Half-Life'' (film), a 2008 independent film by Jennifer Phang * ''Half Life: ...
. Cell turnover rates can be monitored through pulse-chase analysis. Researchers use a combination of pulse-chase analysis and laser ablation electrospray ionization mass spectrometry (LAESI-MS) in order to mechanically increase cell turnover rate. Amino acids were used to label
photosynthetic Photosynthesis ( ) is a Biological system, system of biological processes by which Photoautotrophism, photosynthetic organisms, such as most plants, algae, and cyanobacteria, convert light energy, typically from sunlight, into the chemical ener ...
green algae and
Chlamydomonas reinhardtii ''Chlamydomonas reinhardtii'' is a single-cell green alga about 10 micrometres in diameter that swims with two flagella. It has a cell wall made of hydroxyproline-rich glycoproteins, a large cup-shaped chloroplast, a large pyrenoid, and a ...
cells. Pulse-chase analysis was used to track the isotopic amino acid labels. This was compared to photosynthesis rates in cells, contributing to knowledge of cell life cycle.


Procollagen (Protein)

Procollagen is studied because it plays a role in diseases related to connective tissues. Procollagen interacts with collagen and proteins, and it travels through various
organelle In cell biology, an organelle is a specialized subunit, usually within a cell (biology), cell, that has a specific function. The name ''organelle'' comes from the idea that these structures are parts of cells, as Organ (anatomy), organs are to th ...
s such as the
endoplasmic reticulum The endoplasmic reticulum (ER) is a part of a transportation system of the eukaryote, eukaryotic cell, and has many other important functions such as protein folding. The word endoplasmic means "within the cytoplasm", and reticulum is Latin for ...
and the golgi apparatus. PCA is used to study the formation of procollagen and the disruption of its formation. PCA is done by using non-radioactive cells in order to not potentially disturb the cells' function. This experiment also uses AHA to avoid damage to DNA. Human cells were used to extract procollagen from cells. Collagen was labelled with
fluorescent dyes A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with sev ...
. In addition to staining and pulse-chase labelling, RNA was also isolated from cells to perform RT-qPCR to determine the amount of mRNA and collagen at different points of time.


Viral Inclusions

Pulse-Chase Analysis can be used to determine how viruses replicate and enter the cell. Researchers use Fluorescence Loss After Photoactivation (FLAPh) in order to study
Influenza A virus ''Influenza A virus'' (''Alphainfluenzavirus influenzae'') or IAV is the only species of the genus ''Alphainfluenzavirus'' of the virus family '' Orthomyxoviridae''. It is a pathogen with strains that infect birds and some mammals, as well as c ...
(IAV) in order to study the cell structure during infection. As opposed to
fluorescence recovery after photobleaching Fluorescence recovery after photobleaching (FRAP) is a method for determining the kinetics of diffusion through tissue or cells. It is capable of quantifying the two-dimensional lateral diffusion of a molecularly thin film containing fluorescently ...
(FRAP), FLAPh can determine mobile elements. Viral infections are constantly moving and require a method that can image it while it is moving. FLAPh photobleaches protein components for only short periods of time, making it a suitable method to determine the location of protein and cell components. This was combined with pulse-chase analysis, and researchers used both visual methods to determine the protein decay and location of the virus in the cell through stages of viral infection.


Research


Structure Analysis

PCA can be used to determine the structure of molecules that interact with proteins. This can be done by labelling a protein and analyzing the length of a chase in an experiment, and it can also be done by analyzing amounts concentrations of proteins after a pulse-chase experiment.


= Bacteriophages

= PCA can be used to determine the structure of
bacteriophage A bacteriophage (), also known informally as a phage (), is a virus that infects and replicates within bacteria. The term is derived . Bacteriophages are composed of proteins that Capsid, encapsulate a DNA or RNA genome, and may have structu ...
s. Researchers have used pulse-chase analysis and looked at the delay between the "pulse" and "chase" in order to determine the structure and making of the tail of the bacteriophage T4. In this experiment, amino acids were used to label proteins of the bacteriophage. The phage was made up of 420 proteins, and the position of the tail proteins were determined by the length of the chase. The "chase" of the experiment observed the length of time it took for the protein to assemble in the bacteriophage. The more "inward" in a protein a label has travel, the longer the chase will be. The proteins that were labelled were identified through
gel electrophoresis Gel electrophoresis is an electrophoresis method for separation and analysis of biomacromolecules (DNA, RNA, proteins, etc.) and their fragments, based on their size and charge through a gel. It is used in clinical chemistry to separate ...
. This procedure was repeated until the order of proteins on the bacteriophage tail was determined.


= Macromolecules

= Proteins are studied using PCA to determine how they interact with a larger
macromolecular A macromolecule is a "molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass." Polymers are physi ...
structure. This is done through using an amino acid to start translation. A labelled protein is attached to mRNA, and the protein is followed through translation. After translation has stopped, the protein is chased after translation as well to determine the post-translational behavior. The labels can be used to isolate proteins, and the proteins can be washed and analyzed using immunoprecipitation to identify protein macromolecule complexes and other translational materials.


References

{{reflist Cell imaging Wikipedia Student Program