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Labeling of a protein with a specific dye or tag at defined positions is a critical step in tracing the subtle behavior of the protein and assessing its cellular function. In the past, many strategies have been developed to achieve selective labeling of proteins in living cells. In particular, the site-specific unnatural amino acid (UAA) incorporation technique has gained increasing attention since it enables attachment of various organic probes to a specific position of a protein in a more precise way. Here, BOC Sciences provide labeled proteins with click chemistry reactions and unnatural amino acid reactions.
Click Chemistry, also known as link chemistry, combinbing combinatorial chemistry, aims to quickly and reliably complete the chemical synthesis of various molecules through the splicing of small units. Based on click chemistry, the researchers established bioorthogonal noncanonical amino acid tagging (BONCAT) and fluorescent noncanonical amino acid tagging (FUNCAT) technologies. The use of small molecule dyes to specifically label single amino acid residues has many advantages over labeling methods that introduce fusion fluorescent proteins at the N- or C-terminus of the protein. For example: (1) small molecule non-natural amino acids can be inserted into any exposed site of the protein under conditions that do not affect the activity of the protein; (2) many types of markers can be achieved by gene coding methods for studying protein post-translational modifications, such as glycosylation, acetylation, methylation, ubiquitination, and phosphorylation; (3) Orthogonal labeling methods can regulate protein functions in situ in real time.
Studies have shown that by combining fluorescent non-natural amino acid tags and puromycin tags with proximity ligation assay(PLA) technology, source, redistribution, and turnover of nascent proteins can be monitored in real time. Azide and alkyne are both small molecule chemical groups that do not interfere with the normal spatial folding and positioning of proteins in the body, and therefore do not affect the function of the labeled protein. It is difficult to detect and identify low-abundance nascent proteins using isotope labeling, while click-chemistry-labeled nascent proteins can be easily detected after enrichment and purification. In addition, click chemistry labeled proteins technology can also be used to improve the efficiency of drug binding, providing new ideas for clinical treatment of some diseases.
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