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-Cy7 is a far red-emitting tandem fluorophore that combines phycoerythrin (PE) and Cy7. The donor molecule, PE can be excited by the 488-nm blue, 532-nm green, or 561-nm yellow-green laser and transfers energy to the acceptor molecule, Cy7, which emits light that can be captured with a 780/60 nm bandpass filter. PE-Cy7 has an excitation peak at 565 nm and an emission peak at 778 nm.
SpyCatcher003 (S49C) has a unique cysteine for precise labeling with PE-Cy7 via maleimide chemistry for instance. We have successfully used this PE-Cy7 labelled SpyCatcher003 (S49C) protein to conjugate a recombinant OKT3 antibody that already has a SpyTag. The result PE-Cy7 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-Cy7 molecules to which at least one molecule of SpyCatcher003 (S49C) is bound.
2. Add 3mg PE-Cy7 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.
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