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|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 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
- Target Species
- Protein/peptide tag
- Product Form
- SpyCatcher2 purified recombinant protein - liquid
- Recombinant protein expressed in E. coli and purified by affinity chromatography
- Buffer Solution
- Phosphate buffered saline
- Preservative Stabilisers
- 0.0095% MIT
- ≥90% determined by SDS-PAGE under reducing conditions and visualized by coomassie blue staining
- Approx. Protein Concentrations
- 250 µM (3.9 mg/ml)
- Protein Molecular Weight
- Predicted 15687 Da
- For research purposes only
- 12 months from date of despatch
- This product and/or its use is covered by claims of U.S. patents, and/or pending U.S. and non-U.S. patent applications owned by or under license to Bio-Rad Laboratories, Inc. See bio-rad.com/en-us/trademarks for details.
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.
|Application Name||Verified||Min Dilution||Max Dilution|
- Instructions For Use
- View SpyCatcher Coupling Protocol
Antibody Characterization Reference
Hentrich, C. et al. (2020) Periplasmic Expression of SpyTagged Antibody Fragments Enables Rapid Modular Antibody Assembly
[Epub ahead of print] [Epub ahead of print]
Zakeri, B. et al. (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.
Proc Natl Acad Sci U S A. 109 (12): E690-7. Proc Natl Acad Sci U S A. 109 (12): E690-7.
Keeble, A.H. et al. (2017) Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics.
Angew Chem Int Ed Engl. 56 (52): 16521-16525. Angew Chem Int Ed Engl. 56 (52): 16521-16525.
- Licensed Use
- For in vitro research purposes. Any re-sale in any form or any other commercial application needs a written agreement with Bio-Rad.
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