The most effective way to discover more about proteins is to "tag" them with other molecules that can help provide further clues about how they react. This process is referred to as "protein labeling" and it involves creating a covalent bond between the protein molecule and another such as radioactive isotopes, fluorophores, enzymes, or biotin. The technique implemented will be chosen according to the type of 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.
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.
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.
There are two classes of labeling strategies, in vitro and in vivo. In vitro, refers to cells taken as samples from living beings and studies outside of the actual organism. In these instances, labeling involves the formation of a chemical bond between the tag molecule and the amino acids in the target proteins or nucleic acids.
With the required polymerases, ATP, labeled amino acids and nucleotides, using an enzymatic in vitro method can be effective. Although with many commercial kits designed for in vitro DNA transcription it can be somewhat challenging to get the folding and post-translational modifications and minimal protein length that are needed.
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.
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.
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.
There are two classes of labeling strategies, in vitro and in vivo. In vitro, refers to cells taken as samples from living beings and studies outside of the actual organism. In these instances, labeling involves the formation of a chemical bond between the tag molecule and the amino acids in the target proteins or nucleic acids.
With the required polymerases, ATP, labeled amino acids and nucleotides, using an enzymatic in vitro method can be effective. Although with many commercial kits designed for in vitro DNA transcription it can be somewhat challenging to get the folding and post-translational modifications and minimal protein length that are needed.
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.