Description
SpyCatcher2 (H-SpyC2) is a 15.7 kDa protein that forms a stable covalent isopeptide bond with a second protein that includes a SpyTag. The reaction occurs spontaneously on mixing, is rapid, irreversible, high-yielding and shows good specificity. SpyCatcher2 contains a His6-tag at the N-terminus. To avoid deamidation of asparagine, a N105D mutation was introduced ( Hentrich et al. 2020 ). The reaction is robust at pH 5 to 8, at temperatures from +4°C to +37°C, in various buffer conditions (Ca2 + /Mg2 + not needed) and in the presence of detergents. The reaction also occurs inside cells ( in vivo ). Since the speed of the coupling reaction is concentration dependent, it is recommended to use SpyCatcher2 undiluted. All the different formats of SpyCatcher2 are compatible with Bio-Rad's recombinant HuCAL® antibodies with a SpyTag2 at the C-terminus of the heavy chain. Download SpyCatcher coupling protocol SpyCatcher2 (TZC001) is available conjugated to biotin (TZC001B). It is also available as a modified version, SpyCatcher2-CYS (TZC001CYS), which contains a cysteine residue for site-specific conjugation by the user. SpyCatcher2-CYS can dimerize during storage by formation of a disulfide bond via the free cysteines. Before conjugation to these cysteines, the SpyCatcher2-CYS dimers should be mildly reduced e.g., by addition of 5mM DTT and incubation for 1 hr at room temperature, followed by a fast DTT removal step, e.g., by size exclusion chromatography using PD-10 desalting columns, as DTT can interfere with the subsequent conjugation chemistry. Dialysis is not recommended for DTT removal, as the cysteines can oxidize again during this slow process. View all available Catchers
Storage Information
Store at -20°C only.
Storage in frost-free freezers is not recommended.
This product should be stored undiluted. Avoid repeated freezing and thawing as this may denature the protein. Should this product contain a precipitate we recommend microcentrifugation before use.
