![]() Of key distinction is a reactive moiety or group (indicated in red) that crosslinks the biotinylation reagent to either distinct amino acid functional groups or non-distinct domains available on all amino acids the reactivity of a given reagent is dependent upon the reactive group used. Because of the greater availability of chemical biotinylation reagents and customization, though, this article focuses solely on chemical methods of biotinylation.Īll biotinylation reagents have similar features, as diagrammed below, and variations in these features give biotinylation reagents distinct characteristics that are ideal for different types of experiments. Enzymatic methods require the co-expression of bacterial biotin ligase and an exogenously expressed protein of interest that is modified to carry a biotin acceptor peptide, which provides a more uniform biotinylation than chemical methods and can be cell compartment specific. Chemical methods provide greater flexibility in the type of biotinylation needed than enzymatic approaches and can be performed both in vitro and in vivo. Biocytin differs from biotin by the addition of a lysine group attached to the valeric acid side chain.īiotinylation, also called biotin labeling, is most commonly performed through chemical means, although enzymatic methods are also available. Biocytin can also be used to make trifunctional crosslinking reagents because of the free carboxylate group and α-amine.Ĭomparison of biotin and biocytin. As shown below, biocytin is longer than biotin, which makes the molecule useful in making long-chain biotinylation reagents. This feature allows many useful biotinylation reagents to be created.īiocytin is a derivative of biotin found in serum and urine that has an added lysine group coupled at the ε-amino acid side chain to the valeric acid side chain. Second, as shown in the diagram below, biotin has a valeric acid side chain that is easily derivatized and conjugated to reactive moieties and chemical structures without affecting its avidin-binding function. First, biotin is comparatively smaller than globular proteins, which minimizes any significant interference in many proteins and allows multiple biotin molecules to be conjugated to a single protein for maximum detection by avidin. Proteins that are biotin labeled (i.e., biotinylated) are routinely detected or purified with avidin conjugates in many protein research applications, including the enzyme-linked immunosorbent assay (ELISA), western blot analysis, immunohistochemistry (IHC), immunoprecipitation (IP) and other methods of affinity purification, cell surface labeling and flow cytometry/fluorescence-activated cell sorting (FACS).īesides a strong affinity for avidin, biotin exhibits two characteristics that make the molecule ideal for labeling proteins and macromolecules. The variability of these reagents substantially expand the range of applications for avidin–biotin chemistry. Cleavable or reversible biotinylation reagents enable the specific elution of biotinylated molecules from biotin-binding proteins. Besides functional group specificity, biotinylation reagents are available with different solubility characteristics to focus biotinylation to distinct microenvironments either inside or outside cells. The variety of biotinylation reagents with different functional group specificities is extremely useful, allowing one to choose a reagent that does not inactivate the target macromolecule. Photoreactive biotin compounds that react nonspecifically upon exposure to ultraviolet (UV) light are also available and expand the scope of the molecules that may be biotinylated. Biotinylation reagents are available for targeting specific functional groups or residues, including primary amines, sulfhydryls, carboxyls and carbohydrates. ![]() ![]() Biotinylation is the process of attaching biotin to proteins and other macromolecules. ![]() The biotin–avidin interaction is commonly exploited to detect and/or purify proteins because of the high specificity that these two molecules have for each other. ![]()
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