Description
BiSpyCatcher2 (H-BiSpyC2) is a 29 kDa homodimer of SpyCatcher2 bridged with a flexible linker. Each SpyCatcher2 subunit can form 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. BiSpyCatcher2 contains a His6-tag at the N-terminus. To avoid deamidation of asparagine, a N105D mutation was introduced ( Hentrich et al. 2020 ) for each of the two Catchers. 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 BiSpyCatcher2 undiluted. All the different formats of BiSpyCatcher2 are compatible with Bio-Rad's recombinant HuCAL® antibodies with a SpyTag2 at the C-terminus of the heavy chain. Download BiSpyCatcher coupling protocol BiSpyCatcher2 is available in two modified versions, BiSpyCatcher2-CYS and BiSpyCatcher-CYS3, which contain one or three cysteine residues, respectively, for site-specific conjugation by the user. BiSpyCatcher2 is also available conjugated to biotin and HRP. BiSpyCatcher2-CYS can dimerize by formation of a disulfide bond via the free cysteines. Before conjugation to these cysteines, the BiSpyCatcher2-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. BiSpyCatcher2-CYS3 contains DTT to avoid oligomerization through disulfide bond formation. Immediately before conjugation, to avoid oxidation and disulfide bond formation, carry out a fast DTT removal step, e.g., by size exclusion chromatography. 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.
