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BiSpyCatcher2

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BiSpyCatcher2 thumbnail image 1 BiSpyCatcher2 thumbnail image 2 BiSpyCatcher2 thumbnail image 3 BiSpyCatcher2 thumbnail image 4 BiSpyCatcher2 thumbnail image 5
BiSpyCatcher2 gallery image 1
Schematic image of (A) BiSpyCatcher2, (B) BiSpyCatcher2-CYS, (C) BiSpyCatcher2-CYS3 (D) Fab with SpyTag coupled to BiSpyCatcher2-Cys conjugated to a label via the Cys residue (E) Fab with SpyTag coupled to BiSpyCatcher2-Cys3 conjugated to a label via the Cys residues.
BiSpyCatcher2 gallery image 2
His-BiSpyCatcher2 (TZC002) 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-BiSpyCatcher2 was run in lane 2 and His-BiSpyCatcher2 coupled to a SpyTagged antibody (AbD35759, Fab-F-Spy2-H format) in lane 3. Coupling was done with a 150% molar excess of Fab (25% excess of Fab over SpyCatcher sites) for 1hr at RT.
BiSpyCatcher2 gallery image 3
His-BiSpyCatcher2 (TZC002) coupling kinetic was analyzed on a 4-20% Criterion TGX Stain-Free gel. A SpyTagged Fab (AbD35759, Fab-F-Spy2-H format) was loaded in lane 1 and His-BiSpyCatcher2 in lane 2. Precision Plus Protein Unstained Protein Standard was run in lane 4. In lanes 6-12 the coupling reaction of the Fab (150% molar excess) with His-BiSpyCatcher2 at RT was loaded. The reaction was stopped at the indicated time by addition of 4x Laemmli Sample Buffer. All lanes were loaded with 2 µg protein.
BiSpyCatcher2 gallery image 4
His-BiSpyCatcher2-Cys (TZC002CYS) 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-BiSpyCatcher2-Cys was run in lane 2 and His-BiSpyCatcher2-Cys coupled to a SpyTagged antibody (AbD35759, Fab-F-Spy2-H format) in lane 3. Coupling was done with a 150% molar excess of Fab (25% excess of Fab over SpyCatcher sites) for 1hr at RT.
BiSpyCatcher2 gallery image 5
His-BiSpyCatcher2-Cys3 (TZC002CYS3) 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-BiSpyCatcher2-Cys3 was run in lane 2 and His-BiSpyCatcher2-Cys3 coupled to a SpyTagged antibody (AbD35759, Fab-F-Spy2-H format) in lane 3. Coupling was done with a 150% molar excess of Fab (25% excess of Fab over SpyCatcher sites) for 1hr at RT.

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BiSpyCatcher2

Product Type
Recombinant Protein
Specificity
BiSpyCatcher2

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Product Code Applications Pack Size List Price Your Price Qty
TZC002
Datasheet Datasheet Datasheet
SDS Safety Datasheet SDS
IY 1 mg
List Price Your Price

BiSpyCatcher2 (H-BiSpyC2) is a 29 kDa homodimer of SpyCatcher2 (TZC001) 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 (TZC002) is available in two modified versions, BiSpyCatcher2-CYS (TZC002CYS) and BiSpyCatcher-CYS3 (TZC002CYS3), which contain one or three cysteine residues, respectively, for site-specific conjugation by the user.
BiSpyCatcher2 (TZC002) is also available conjugated to biotin (TZC002B) and HRP (TZC002P).

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.

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Target Species
Protein/peptide tag
Product Form
BiSpyCatcher2 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
0.1% ProClin 950
Purity
≥90% determined by SDS-PAGE under reducing conditions and visualized by coomassie blue staining
Approx. Protein Concentrations
80 µM (2.3 mg/ml)
Protein Molecular Weight
Predicted 29018 Da
Regulatory
For research purposes only
Guarantee
12 months from date of despatch
Acknowledgements
ProClin is a trademark of The Dow Chemical Company (“Dow”) or an affiliated company of Dow
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 BiSpyCatcher 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
anti SpyCatcher HCA379 E F WB 0.1 mg
List Price Your Price
Description anti SpyCatcher
BiSpyCatcher2:Biotin TZC002B IY 1 mg
List Price Your Price
Description BiSpyCatcher2:Biotin
BiSpyCatcher2-CYS TZC002CYS IY 1 mg
List Price Your Price
Description BiSpyCatcher2-CYS
BiSpyCatcher2-CYS3 TZC002CYS3 IY 1 mg
List Price Your Price
Description BiSpyCatcher2-CYS3
BiSpyCatcher2:HRP TZC002P IY 1 mg
List Price Your Price
Description BiSpyCatcher2:HRP

Antibody Characterization Reference

  1. Hentrich, C. et al. (2021) Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly.
    Cell Chem Biol. 28 (6): 813-824.e6. Cell Chem Biol. 28 (6): 813-824.e6.
  2. 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.
  3. 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.

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.

TZC002

156817 160509

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