Scientists can learn more about specific proteins by "labeling" them, that is attaching a molecule such as biotin, enzymes, radioactive isotopes, or fluorophores covalently to enable more efficient detection or purification of the protein itself. Protein labeling is an important process used in research of which there are several methods and strategies that can be employed, depending on the nature of the application.
The water soluble B-vitamin and coenzyme biotin makes an ideal label marker because of its natural efficacy for forming strong bonds with various proteins and nucleotides. It is smaller than its enzymatic counterparts, which causes less interference with the protein's functions. "Biotinylation" refers to tagging nucleotides and proteins with biotin on an enzymatic or chemical level. The solubility of the molecules can increase or decrease as a result.
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.
Enzymes are versatile in nature, and many of them possess a long shelf-life and can also be used as effective tags for protein detection in certain cells and tissues. They are larger molecules than biotin, and normally require that a substrate be combined with their use in order to produce a detectable result in the form of a fluorescent, chromogenic, or chemiluminescent signal. Commonly used enzymes include glucose oxidase, alkaline phosphatase, and horseradish peroxidase.
Fluorescent probes or fluorophores give off a luminescent signal in response to light. They don't require the use of a reagent and they are quite versatile, which lend themselves to applications such as determining the location, activation, and formation of proteins and monitoring in vivo biological processes. The three types of fluorophores are quantum dots, biological fluorophores, and organic dyes. Specialized equipment including cell sorters, flow cytometers, and fluorescence plate-readers and microscopes are used to detect these probes.
Labeling strategies can be classified as either in vivo or in vitro. The latter approach involves obtaining a sampling of cells from a living organism and studying them in a laboratory. A tag molecule is bonded with the amino acids contained in the specific proteins or nucleic acids being evaluated.
Some limitations exist when using commercial kits for in vitro DNA transcription, in that it may be difficult to obtain the appropriate protein length and post-translational and folding modifications. However with the necessary ATP and polymerases, and labeled nucleotides, and amino acids, these enzymatic in vitro approaches can still be useful.
Living organisms, which are usually lab animals are used for in vivo methods. Termed "metabolic labeling", this approach involves the culturing of cellular proteins and nucleic acids with certain labeled nucleotides and amino acids. Using this technique promotes consistency and is helpful in the further purification of proteins. The primary drawback is that appropriate reagents are few, and some labels may be toxic if used, so precautions are necessary.
The water soluble B-vitamin and coenzyme biotin makes an ideal label marker because of its natural efficacy for forming strong bonds with various proteins and nucleotides. It is smaller than its enzymatic counterparts, which causes less interference with the protein's functions. "Biotinylation" refers to tagging nucleotides and proteins with biotin on an enzymatic or chemical level. The solubility of the molecules can increase or decrease as a result.
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.
Enzymes are versatile in nature, and many of them possess a long shelf-life and can also be used as effective tags for protein detection in certain cells and tissues. They are larger molecules than biotin, and normally require that a substrate be combined with their use in order to produce a detectable result in the form of a fluorescent, chromogenic, or chemiluminescent signal. Commonly used enzymes include glucose oxidase, alkaline phosphatase, and horseradish peroxidase.
Fluorescent probes or fluorophores give off a luminescent signal in response to light. They don't require the use of a reagent and they are quite versatile, which lend themselves to applications such as determining the location, activation, and formation of proteins and monitoring in vivo biological processes. The three types of fluorophores are quantum dots, biological fluorophores, and organic dyes. Specialized equipment including cell sorters, flow cytometers, and fluorescence plate-readers and microscopes are used to detect these probes.
Labeling strategies can be classified as either in vivo or in vitro. The latter approach involves obtaining a sampling of cells from a living organism and studying them in a laboratory. A tag molecule is bonded with the amino acids contained in the specific proteins or nucleic acids being evaluated.
Some limitations exist when using commercial kits for in vitro DNA transcription, in that it may be difficult to obtain the appropriate protein length and post-translational and folding modifications. However with the necessary ATP and polymerases, and labeled nucleotides, and amino acids, these enzymatic in vitro approaches can still be useful.
Living organisms, which are usually lab animals are used for in vivo methods. Termed "metabolic labeling", this approach involves the culturing of cellular proteins and nucleic acids with certain labeled nucleotides and amino acids. Using this technique promotes consistency and is helpful in the further purification of proteins. The primary drawback is that appropriate reagents are few, and some labels may be toxic if used, so precautions are necessary.
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