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.
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.
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.
Read more about Protein Labeling Techniques And Applications.
Author: Arline BradleyThis author has published 67 articles so far.