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|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
- Target Species
- Protein/peptide tag
- Product Form
- SpyCatcher3 purified recombinant protein, with an engineered cysteine residue; used for site-specific chemical conjugation to a label of choice - liquid
- Recombinant protein expressed in E. coli and purified by affinity chromatography
- Buffer Solution
- Phosphate buffered saline
- Preservative Stabilisers
- 0.0095% MIT
5 mM EDTA
- ≥90% determined by SDS-PAGE under reducing conditions and visualized by coomassie blue staining
- Approx. Protein Concentrations
- 455 µM (7 mg/ml)
- Protein Molecular Weight
- Predicted 15368 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. (2019) Approaching infinite affinity through engineering of peptide-protein interaction.
Proc Natl Acad Sci U S A. Dec 10 [Epub ahead of print]. Proc Natl Acad Sci U S A. Dec 10 [Epub ahead of print].
- 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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