SpyCatcher3

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TZC025CYS
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SDS Safety Datasheet SDS
IY 1 mg loader
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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.

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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
Preparation
Recombinant protein expressed in E. coli and purified by affinity chromatography
Buffer Solution
Phosphate buffered saline
Preservative Stabilisers
0.0095% MIT
5 mM EDTA
Purity
≥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
Regulatory
For research purposes only
Guarantee
12 months from date of despatch
Acknowledgements
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.

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.

This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.
Application Name Verified Min Dilution Max Dilution
Immunoassay
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.
Instructions For Use
View SpyCatcher Coupling Protocol

Description Product Code Applications Pack Size List Price Your Price Quantity
SpyTag3 Peptide BLP086 IY 1 mg loader
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loader
Description SpyTag3 Peptide
SpyCatcher3 TZC025 IY 1 mg loader
List Price Your Price
loader
Description SpyCatcher3

Antibody Characterization Reference

  1. Hentrich, C. et al. (2020) Periplasmic Expression of SpyTagged Antibody Fragments Enables Rapid Modular Antibody Assembly
    [Epub ahead of print] [Epub ahead of print]
  2. 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.
  3. 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.