Description
SpyCatcher3 (H-SpyC3) is a 15.2 kDa protein that forms a stable covalent isopeptide bond with a second protein that includes a SpyTag ( Keeble et al. 2019 ). The reaction occurs spontaneously on mixing, is rapid, irreversible, high-yielding and shows good specificity. SpyCatcher3 contains a His6-tag at the N-terminus. 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 SpyCatcher3 undiluted. All the different formats of SpyCatcher3 are compatible with Bio-Rad's recombinant HuCAL antibodies with a SpyTag2 at the C-terminus of the heavy chain. SpyCatcher3 couples faster to SpyTag than the earlier SpyCatcher versions. At neat concentrations the difference in speed between SpyCatcher3 and SpyCatcher2 is negligible but with diluted SpyTag and/or SpyCatcher using SpyCatcher3 shows some benefits. Therefore, Spycatcher3 is recommended for immobilization on plates or beads to bind a capture antibody or other proteins with a SpyTag. Download SpyCatcher coupling protocol SpyCatcher3 (TZC025) is available as a modified version, SpyCatcher3-CYS (TZC025CYS), which contains a cysteine residue for site-specific conjugation by the user SpyCatcher3-CYS can dimerize by formation of a disulfide bond via the free cysteines. Before conjugation to these cysteines, the SpyCatcher3-CYS must be 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, as DTT can interfere with the 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.
