生物素(Biotin)为B族维生素之一,又称维生素H、维生素B7、辅酶R(Coenzyme R)等。生物素是一种水溶性维生素,属于B族维生素的一种。
生物素化,也称为生物素标记,是将生物素共价连接到生物分子(如蛋白质、抗体、肽、寡核苷酸和其他大分子)的过程。该反应是快速、特异性的,并且由于生物素体积小 (MW = 244.31) 而不太可能干扰生物分子的自然功能。
生物素与链霉亲和素和亲和素特异性结合,形成具有强亲和力 (Kd, ~ 10-14 mol/L) 和快速接通速率的复合物。即使在高/低 pH 值、高温、高盐浓度等恶劣条件下,复合物也非常稳定。
生物素-亲和素和链霉亲和素系统广泛用于靶抗原/细胞的检测和分离。这些应用包括免疫测定(ELISA 和 Western 是常规应用)、亲和色谱、沉降测定、超分子构建、靶向癌细胞进行药物递送等。最近,基于生物素-(strept)亲和素相互作用和磁珠的蛋白质/细胞分离的应用需求不断增加。
Broadpharm知识课堂什么是生物素化?What is Biotinylation?
Biotinylation, also known as biotin labeling, is the process of covalently attaching biotin(s) to biomolecules: such as proteins, antibodies, peptide, oligonucleotide, and other macromolecules. The reaction is rapid, specific and is unlikely to disturb the natural function of the biomolecules due to the small size of biotin (MW = 244.31).
Biotin specifically binds to streptavidin and avidin to form a complex with an extremely high affinity (Kd, ~ 10-14 mol/L) and fast on-rate. The complexes are very stable under even extreme conditions such as high/low pH, high temperature, high salt concentrations, etc.
Biotin-avidin and streptavidin systems are widely used in detection and separation of target antigens/cells. These applications include immunoassays (ELISAs and Westerns being the most popular applications), affinity chromatography, pull-down assays, supramolecular construction, targeting of cancer cells for drug delivery, and many others. Recently, there are increasing application demands for protein/cell separation based on biotin-(strept)avidin interaction and magnetic beads.
For biotinylation chemistry, the most common reactions involve amines with biotin-NHS ester and click chemistry of azide with an alkyne (e.g. DBCO), as shown in Figure 2.
The selection of biotinylation reagent should consider a few factors: target functional group, water solubility, cell membrane permeability, cleavability, and length of the reagent.
The functional groups can be click chemistry reactive, amine reactive, carbonyl reactive, carboxyl reactive, and sulfhydryl reactive (Figure 3). There are also reversible and cleavable biotinylation reagents to help with the specific elution of biotinylated proteins.
Pegylated biotin reagents are particularly attractive due to their water solubility, no toxicity, and low immunogenic properties. Monodispersed PEGs have a well-defined chain length, allowing for specific biotin-based complexes to be designed and studied.
In addition, BroadPharm provides desthiobiotin products for special application, such as affinity purification. Desthiobiotin is a modified form of biotin that binds less tightly to avidin and streptavidin than biotin while still providing excellent specificity. Unlike biomolecules that are labeled with biotin, proteins and other targets labeled with desthiobiotin can be eluted under a soft, mild elute conditions to avoid denaturing the protein of interest.
BroadPharm is a leader of biotinylation reagents that help advance our customer's research, and offers a variety of pegylated and non-pegylated biotinylation reagents to meet your requirement.
1. Cull and Schatz, "Biotinylation of proteins in vivo and in vitro using small peptide tags", Methods in Enzymology, 326, (2000): 430-440
2. Minde, et al., "Biotin proximity tagging favours unfolded proteins and enables the study of intrinsically disordered regions", Communications Biology, 3, 38, (2020): 1-13