Tagging or labeling specific proteins allows researchers to detect and purify these molecules and gain a better understanding of how they react. The process of protein labeling consists of covalently attaching another molecule, such as an enzyme, biotin, fluorophore, or radioactive isotope to the protein for the purpose of monitoring its behavior. The type of labeling used depends on the particular application.
Biotin is a naturally-derived coenzyme and B-vitamin which is an ideal label due to its ability to bind strongly to numerous proteins. Smaller than enzyme labels, it is unlikely to disrupt normal functioning of proteins. This process of implementing biotin as a protein or nucleotide label is known as "biotinylation" and it can be done on both a chemical and enzymatic level and it can increase or decrease the solubility of the proteins.
Sometimes enzymes are the molecules of interest, in which case a chemical reagent known as an "active site probe". These electrophilic probes are used for the purpose of identifying, profiling, and enriching classes of enzymes such as phosphatases, kinases, and GTPases, to name a few. They can also detect when the action of the targeted enzymes has been inhibited by other molecules.
When enzymes are chosen as the labels for proteins, it is usually necessary to also add a substrate with them so that a notable response will be produced either in the form of a chromogenic, chemiluminescent, or fluorescent signal. Some of the enzymes used for this purpose are horseradish peroxidase, glucose oxidase, and alkaline phosphatase.
Fluorophores are fluorescent probes are those which respond to light, producing a visibly luminescent signal. No additional reagent is needed with these tags and their versatility is well suited for applications such as monitoring in vivo biological processes, and in the detection of protein location, formation, and activation. There are three types; biological fluorophores, organic dyes, and quantum dots. It is necessary to implement special equipment to detect these probes however, such as fluorescence microscopes or plate-readers, cell sorters, and flow cytometers.
Labeling strategies may be classified as either in vitro or in vivo. The former involves taking samples of cells from a living host organism and conducting studies on them in a laboratory. Target proteins and nucleic acids are "labeled" when the tag molecule binds with their amino acids.
Commercial kits are available for enzymatic, in vitro DNA transcription, but there are some limitations on their effectiveness as it can be challenging to obtain a suitable protein length as well as folding and post-translational modifications. They can still be of some use however, provided the correct amino acids, polymerases, ATP, and labeled nucleotides are utilized.
In vivo techniques are carried out in living organisms, usually animals, and are often referred to as "metabolic labeling". They consist of culturing the proteins and nucleic acids in a cell or group of cells with specific labeled amino acids and nucleotides. This process facilitates the purification of proteins and helps ensure consistency. However, the suitable reagents are limited and some labels may be toxic, so caution must be exercised.
Biotin is a naturally-derived coenzyme and B-vitamin which is an ideal label due to its ability to bind strongly to numerous proteins. Smaller than enzyme labels, it is unlikely to disrupt normal functioning of proteins. This process of implementing biotin as a protein or nucleotide label is known as "biotinylation" and it can be done on both a chemical and enzymatic level and it can increase or decrease the solubility of the proteins.
Sometimes enzymes are the molecules of interest, in which case a chemical reagent known as an "active site probe". These electrophilic probes are used for the purpose of identifying, profiling, and enriching classes of enzymes such as phosphatases, kinases, and GTPases, to name a few. They can also detect when the action of the targeted enzymes has been inhibited by other molecules.
When enzymes are chosen as the labels for proteins, it is usually necessary to also add a substrate with them so that a notable response will be produced either in the form of a chromogenic, chemiluminescent, or fluorescent signal. Some of the enzymes used for this purpose are horseradish peroxidase, glucose oxidase, and alkaline phosphatase.
Fluorophores are fluorescent probes are those which respond to light, producing a visibly luminescent signal. No additional reagent is needed with these tags and their versatility is well suited for applications such as monitoring in vivo biological processes, and in the detection of protein location, formation, and activation. There are three types; biological fluorophores, organic dyes, and quantum dots. It is necessary to implement special equipment to detect these probes however, such as fluorescence microscopes or plate-readers, cell sorters, and flow cytometers.
Labeling strategies may be classified as either in vitro or in vivo. The former involves taking samples of cells from a living host organism and conducting studies on them in a laboratory. Target proteins and nucleic acids are "labeled" when the tag molecule binds with their amino acids.
Commercial kits are available for enzymatic, in vitro DNA transcription, but there are some limitations on their effectiveness as it can be challenging to obtain a suitable protein length as well as folding and post-translational modifications. They can still be of some use however, provided the correct amino acids, polymerases, ATP, and labeled nucleotides are utilized.
In vivo techniques are carried out in living organisms, usually animals, and are often referred to as "metabolic labeling". They consist of culturing the proteins and nucleic acids in a cell or group of cells with specific labeled amino acids and nucleotides. This process facilitates the purification of proteins and helps ensure consistency. However, the suitable reagents are limited and some labels may be toxic, so caution must be exercised.
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