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SpyCatcher2

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SpyCatcher2 thumbnail image 1 SpyCatcher2 thumbnail image 2 SpyCatcher2 thumbnail image 3 SpyCatcher2 thumbnail image 4
SpyCatcher2 gallery image 1
Schematic image of (A) SpyCatcher2, (B) SpyCatcher2-CYS, (C) Fab with SpyTag coupled to SpyCatcher conjugated to a label via the Cys residue.
SpyCatcher2 gallery image 2
His-SpyCatcher2 (TZC001) was analyzed on a 4-20% Criterion TGX Stain-Free gel. Precision Plus Protein Unstained Protein Standard was loaded in lanes 1 & 4. Uncoupled His-SpyCatcher2 was run in lane 2 and His-SpyCatcher2 coupled to a SpyTagged antibody (AbD35759, Fab-F-Spy2-H format) in lane 3. Coupling was done with a 25% molar excess of SpyCatcher2 for 1 hr at RT. All lanes were loaded with 1 μg protein.
SpyCatcher2 gallery image 3
His-SpyCatcher2 (TZC001) coupling kinetic was analyzed on an AnyKD Criterion TGX Stain-Free gel. A SpyTagged Fab (AbD35759, Fab-F-Spy2-H format) was loaded in lane 1 and His-SpyCatcher2 in lane 2. Precision Plus Protein Unstained Protein Standard was run in lane 4. In lanes 6-12 the coupling reaction of the Fab with a 25% molar excess of His-SpyCatcher2 at RT was loaded. The reaction was stopped at the indicated time by addition of 4x Laemmli Sample Buffer. All lanes were loaded with 3 μg protein.
SpyCatcher2 gallery image 4
His-SpyCatcher2-Cys (TZC001CYS) was analyzed on a 4-20% Criterion TGX Stain-Free gel. Precision Plus Protein Unstained Protein Standard was loaded in lanes 1 & 4. Uncoupled His-SpyCatcher2-Cys was run in lane 2 and His-SpyCatcher2-Cys coupled to a SpyTagged antibody (AbD35759, Fab-F-Spy2-H format) in lane 3. Coupling was done with a 25% molar excess of SpyCatcher2 for 1 hr at RT. All lanes were loaded with 2 μg protein.

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SpyCatcher2

Product Type
Recombinant Protein
Specificity
SpyCatcher2

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Product Code Applications Pack Size List Price Your Price Qty
TZC001
Datasheet Datasheet Datasheet
SDS Safety Datasheet SDS
IY 1 mg loader
List Price Your Price
loader

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 conjugated to biotin (TZC001B). It is also 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.

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Target Species
Protein/peptide tag
Product Form
SpyCatcher2 purified recombinant protein - liquid
Preparation
Recombinant protein expressed in E. coli and purified by affinity chromatography
Source
E.coli
Buffer Solution
Phosphate buffered saline
Preservative Stabilisers
0.0095% MIT
Purity
≥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
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.
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.

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
TZC001
View SpyCatcher Coupling Protocol

Description Product Code Applications Pack Size List Price Your Price Quantity
SpyTag3 Peptide BLP086 IY 1 mg loader
List Price Your Price
loader
Description SpyTag3 Peptide
SpyCatcher2:Biotin TZC001B IY 1 mg loader
List Price Your Price
loader
Description SpyCatcher2:Biotin
SpyCatcher2-CYS TZC001CYS IY 1 mg loader
List Price Your Price
loader
Description SpyCatcher2-CYS

Source Reference

  1. 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.

References for SpyCatcher2

  1. Keeble, A.H. et al. (2017) Evolving Accelerated Amidation by SpyTag/SpyCatcher to Analyze Membrane Dynamics.
    Angew Chem Int Ed Engl. 56 (52): 16521-5.
  2. Hentrich, C. et al. (2021) Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly.
    Cell Chem Biol. 28 (6): 813-824.e6.

TZC001

156819 161627 164878 167145

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Please Note: All Products are "FOR RESEARCH PURPOSES ONLY"

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