SpyCatcher antibody | AbD41909kg
A microtiter plate was coated overnight with SpyCatcher1, SpyCatcher2 (TZC001) or SpyCatcher3 (TZC025) uncoupled or coupled to SpyTag3 peptide, at a concentration of 1 μg/ml. After washing and blocking with PBST + 5% BSA, detection was performed using Anti-SpyCatcher Antibody, clone AbD41909 in monovalent Fab format, titrated to the given concentrations in PBST followed by an HRP conjugated anti-DYKDDDDK tag antibody in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate.
A microtiter plate was coated overnight with Anti-SpyCatcher Antibody, clone AbD41909kg (HCA379) at a concentration of 1 μg/ml. After washing and blocking with PBST + 5% BSA, five variants of SpyCatcher were added: biotin conjugated SpyCatcher1, SpyCatcher2, or SpyCatcher3, or biotin conjugated SpyCatcher2 coupled to a SpyTagged Fab antibody, HuCAL Fab-F-Spy2-H Negative Control Antibody (TZA003), or to SpyTagged maltose binding protein (MBP-SpyTag2). Detection was performed using HRP conjugated Neutravidin in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate.
5 ng of SpyCatcher1, SpyCatcher2 (TZC001) or SpyCatcher3 (TZC025) (lanes 2-4) were run on a 4-20% Criterion TGX Stain-Free Protein Gel (Bio-Rad #5678095) under denaturing conditions; Precision Plus Protein All Blue Prestained Protein Standards (Bio-Rad #1610373) was run in lane 1. Proteins were transferred to a 0.2 μm PVDF membrane (Bio-Rad #1704157) and detection was performed using Anti-SpyCatcher Antibody, clone AbD41909kg (HCA379) diluted 1:300 in TBST + 1% BSA, followed by Goat Anti-Rabbit IgG (H/L):HRP Antibody (STAR124P) and QuantaBlu Fluorogenic Peroxidase. *Presumed dimer of SpyCatcher1.
1 ng of SpyCatcher1 (lanes 2 and 5), SpyCatcher2 (TZC001) (lanes 3 and 6) and SpyCatcher3 (TZC025) (lanes 4 and 7) coupled either to a SpyTagged Fab antibody, HuCAL Fab-F-Spy2-H Negative Control Antibody (TZA003), or to SpyTagged maltose binding protein (MBP-SpyTag2) were run on a 4-20% Criterion TGX Stain-Free Protein Gel (Bio-Rad #5678095) under denaturing conditions; Precision Plus Protein All Blue Prestained Protein Standards (Bio-Rad # 1610373) was run in lane 1. Proteins were transferred to a 0.2 μm PVDF membrane (Bio-Rad #1704157) and detection was performed using Anti-SpyCatcher Antibody, clone AbD41909kg (HCA379) diluted 1:300 in TBST + 1% BSA, followed by Goat Anti-Rabbit IgG (H/L):HRP Antibody (STAR124P) and QuantaBlu Fluorogenic Peroxidase.
1 ng (lane 2), 0.1 ng (lane 3), 0.01 ng (lane 4) of SpyCatcher3 (TZC025) coupled to a SpyTagged Fab antibody, HuCAL Fab-F-Spy2-H Negative Control Antibody (TZA003) was spiked into E. coli lysate and run on a 4-20% Criterion TGX Stain-Free Protein Gel (Bio-Rad #5678095) under denaturing conditions; Precision Plus Protein All Blue Prestained Protein Standards (Bio-Rad #1610373) was run in lane 1. Proteins were transferred to a 0.2 μm PVDF membrane (Bio-Rad #1704157) and detection was performed using Anti-SpyCatcher Antibody, clone AbD41909kg (HCA379) diluted 1:10,000 in TBST + 1% BSA, followed by Goat Anti-Rabbit IgG (H/L):HRP Antibody (STAR124P) and QuantaBlu Fluorogenic Peroxidase.
Jurkat cells were incubated with either an uncoupled Anti-Human CD45 SpyTagged Fab Antibody (grey) or a Anti-Human CD45 SpyTagged Fab Antibody coupled to SpyCatcher2 (TZC001, green) or BiSpyCatcher2 (TZC002, blue) at 50nM in 3% FBS-PBS. After washing with 3% FBS-PBS, detection was performed using 1:2 serial dilution of the Anti-SpyCatcher Antibody, clone AbD41909kg (HCA379) followed by an AlexaFluor®647 conjugated Anti-Rabbit H/L IgG Antibody used at 1:400 in 3% FBS-PBS. A control SpyTagged Fab coupled to SpyCatcher2 (TZC001, red) or BiSpyCatcher2 (TZC002, dark blue) alone were used as negative control.
Jurkat cells were incubated with a Anti-Human CD45 SpyTagged Fab Antibody (AbD40122ad) coupled to either SpyCatcher2 (TZC001; A, green) or BiSpyCatcher2 (TZC002; B, blue) at 50nM in 3% FBS-PBS. After washing in 3% FBS-PBS, detection was performed using the Anti-SpyCatcher Antibody, clone AbD41909kg (HCA379) followed by an AlexaFluor 647 conjugated Anti-Rabbit H/L IgG Antibody used at 1:400 in 3% FBS-PBS. The SpyTagged Fab AbD35759 coupled to either SpyCatcher2 (TZC001; A, red) or BiSpyCatcher2 (TZC002; B, red) was used as a negative control.
anti SpyCatcher
- Product Type
- Monoclonal Antibody
- Clone
- AbD41909kg
- Isotype
- IgG
- Specificity
- SpyCatcher
Antibody Quality and Performance Guarantee
| Anti-SpyCatcher Antibody, clone AbD41909kg is a chimeric antibody comprising a human Fab portion and a rabbit Fc domain, which specifically binds to SpyCatcher protein with high affinity. SpyCatcher is used for protein ligation in combination with the SpyTag (Zakeri et al. 2012). SpyCatcher is derived from the second immunoglobulin-like collagen adhesin domain (CnaB2) from the fibronectin binding protein (FbaB) of Streptococcus pyogenes. Anti-SpyCatcher Antibody binds to the original SpyCatcher as well as the optimized derivatives SpyCatcher2 (Keeble et al. 2017) and SpyCatcher3 (Keeble et al. 2019). It recognizes the SpyCatcher variants in uncoupled form as well as when coupled to the SpyTag peptide or to fusion proteins with C-terminally fused SpyTag. N-terminal fusions were not tested. View a summary of SpyCatcher products |
|
- Target Species
- Synthetic Peptide
- Product Form
- Recombinant chimeric human/rabbit IgG antibody (lambda light chain) selected from the HuCAL phage display library and expressed in a human cell line. In this antibody, the VH-CH1 regions and light chains are human while the Fc region is derived from rabbit IgG. This antibody is supplied as a liquid.
- Preparation
- Purified IgG prepared by affinity chromatography on Protein A
- Source
- HKB-11
- Buffer Solution
- Phosphate buffered saline
- Preservative Stabilisers
- 0.09% Sodium Azide (NaN3)
- Immunogen
- SpyCatcher
- Approx. Protein Concentrations
- Antibody concentration 1.0 mg/ml
- 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.
This product is shipped at ambient temperature. It is recommended to aliquot and store at -20°C on receipt. When thawed, aliquot the sample as needed. Keep aliquots at 2-8°C for short term use (up to 4 weeks) and store the remaining aliquots at -20°C.
Avoid repeated freezing and thawing as this may denature the antibody. Storage in frost-free freezers is not recommended.
Avoid repeated freezing and thawing as this may denature the antibody. Storage in frost-free freezers is not recommended.
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 |
|---|---|---|---|
| ELISA |  |
1/10,000 | 1/20,000 |
| Flow Cytometry | |
1/100 | 1/1,000 |
| Western Blotting | |
1/10,000 | 1/20,000 |
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.
- Technical Advice
- For further information about recombinant antibodies incorporating SpyTag technology visit SpyTag and SpyCatcher Products.
- Flow Cytometry
- Use 10μl of the suggested working dilution to label 1x106 cells in 100μl
| Description | Product Code | Applications | Pack Size | List Price | Your Price | Quantity | |
|---|---|---|---|---|---|---|---|
| Goat anti Rabbit IgG (H/L):HRP | STAR124P | C E WB | 1 mg |
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| Description | Goat anti Rabbit IgG (H/L):HRP | ||||||
| Description | Product Code | Applications | Pack Size | List Price | Your Price | Quantity | |
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| Hispec Assay Diluent | BUF049A | E IY | 50 ml | Log in | |||
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| Description | Hispec Assay Diluent | ||||||
| SpyCatcher2 | TZC001 | IY | 1 mg |
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| Description | SpyCatcher2 | ||||||
| SpyCatcher2-CYS | TZC001CYS | IY | 1 mg |
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| Description | SpyCatcher2-CYS | ||||||
| BiSpyCatcher2 | TZC002 | IY | 1 mg |
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| Description | BiSpyCatcher2 | ||||||
| BiSpyCatcher2-CYS | TZC002CYS | IY | 1 mg |
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| Description | BiSpyCatcher2-CYS | ||||||
| BiSpyCatcher2-CYS3 | TZC002CYS3 | IY | 1 mg |
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| Description | BiSpyCatcher2-CYS3 | ||||||
| Human IgG1-FcSpyCatcher3 | TZC009 | IY | 1 mg |
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| Description | Human IgG1-FcSpyCatcher3 | ||||||
| Mouse IgG2a-FcSpyCatcher3 | TZC012 | IY | 0.5 mg |
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| Description | Mouse IgG2a-FcSpyCatcher3 | ||||||
| Rabbit IgG-FcSpyCatcher3 | TZC013 | IY | 0.5 mg |
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| Description | Rabbit IgG-FcSpyCatcher3 | ||||||
| Human IgG2-FcSpyCatcher3 | TZC016 | IY | 0.5 mg |
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| Description | Human IgG2-FcSpyCatcher3 | ||||||
| Human IgG3-FcSpyCatcher3 | TZC017 | IY | 0.5 mg |
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| Description | Human IgG3-FcSpyCatcher3 | ||||||
| Human IgG4-FcSpyCatcher3 | TZC018 | IY | 0.5 mg |
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| Description | Human IgG4-FcSpyCatcher3 | ||||||
| Human IgG4-Pro-FcSpyCatcher3 | TZC019 | IY | 0.5 mg |
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| Description | Human IgG4-Pro-FcSpyCatcher3 | ||||||
| Human IgA-FcSpyCatcher3 | TZC020 | IY | 0.5 mg |
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| Description | Human IgA-FcSpyCatcher3 | ||||||
| SpyCatcher3 | TZC025 | IY | 1 mg |
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| Description | SpyCatcher3 | ||||||
| SpyCatcher3-CYS | TZC025CYS | IY | 1 mg |
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| Description | SpyCatcher3-CYS | ||||||
HCA379
160403
162794
166597
169426
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