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文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
The SpyTag/SpyCatcher system is a convenient protein coupling tool for irreversible peptide-protein ligation. It is ideal for binding, labeling, immobilization and creating new kinds of protein architectures.
Peptide interaction with proteins is usually weak. SpyTag is a genetically encoded peptide (SpyTag003 sequence RGVPHIVMVDAYKRYK) that forms a spontaneous amide bond upon binding its genetically encoded partner SpyCatcher. SpyTag003 reacts with SpyCatcher under a wide range of conditions.
PE-Cy5 is a red-emitting tandem fluorophore that combines phycoerythrin (PE) and Cy5. The donor molecule, PE can be excited by the 488-nm blue, 532-nm green, or 561-nm yellow-green laser and and transfers energy to the acceptor molecule, Cy5. PE-Cy5 has an excitation peak at 496 nm and an emission peak at 667 nm.
SpyCatcher003 (S49C) has a unique cysteine for precise labeling with PE-Cy5 via maleimide chemistry for instance. We have successfully used this PE-Cy5 labelled SpyCatcher003 (S49C) protein to conjugate a recombinant OKT3 antibody that already has a SpyTag. The result PE-Cy5 labelled OKT3 antibody was successfully used for flow cytometry.
Guideline for Use
1. The concentration is determined by absorption = 82 at 565 nm for a 1% solution for only those PE-Cy5 molecules to which at least one molecule of SpyCatcher003 (S49C) is bound.
2. Add 3mg PE-Cy5 labelled SpyCatcher003 (S49C) protein per mg of Spytag modified antibody. Mix and incubate for 2 hours at room temperature or overnight at 4°C. The labeled antibody can be directly tested by flow cytometry. The amount of protein can be scaled proportionally.
Flow cytometry analysis of CD3 expression by human peripheral blood lymphocytes (right panel) compared to an isotype control (left panel). Cells were stained with Recombinant CD3 Antibody (PE-Cy5) (rp218684) prepared using the LumiDye™ PE-Cy5-labeled SpyCatcher (rp218684) at 5μL/test for 1 hour at 4°C. Left panel - Mouse IgG (PE-Cy5) (Ab190078).
Flow cytometry analysis of CD3 expression by human peripheral blood lymphocytes (red) compared to an isotype control (blue). Cells were stained with Recombinant CD3 Antibody (PE-Cy5) (rp218684) prepared using the LumiDye™ PE-Cy5-labeled SpyCatcher (rp218684) at 5μL/test for 1 hour at 4°C. Blue - Mouse IgG (PE-Cy5) (Ab190078). Black - Unlabelled control, cells without incubation with primary antibody.
ZJ26F0332449
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