LumiDye™ PE-Cy5-labeled SpyCatcher

Cat. No.: rp218684

GRADE & PURITY Carrier Free, Validated, for conjugation, 5mg/mL

Accession #

QGX07219.1

Protein Tag

N-His

Expression system

E. coli

Bioactivity

SpyTag/SpyCatcher Protein Coupling

Overview

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.

Specifications

Product Name

LumiDye™ PE-Cy5-labeled SpyCatcher

Synonyms

SpyCatcher003 (S49C) Protein | SpyCatcher | SpyCatcher003, S49C | SpyCatcher-PE-Cy5

Grade

Carrier Free, for conjugation, Validated

Specifications & Purity

Carrier Free, Validated, for conjugation, 5mg/mL

Bioactivity

SpyTag/SpyCatcher Protein Coupling

Amino Acids

1-144 aa（S49C)

Sequence

MSYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDCSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSSGS​​

Conjugation

PE-Cy5

Excitation (nm)

Blue Laser(488nm), Green Laser(532nm), Yellow-Green Laser(561nm)

Emission(nm)

670nm

Ex/Em(nm)

Accession #

QGX07219.1

Molecule Type

Protein

Storage and Shipping

Concentration

5mg/mL

Storage

Store at 2-8°C,Protected from light,Do not freeze

Shipped In

Wet ice,Do not freeze

Stability And Storage

This product can be stored at 2-8°C for 12 months. Please protected from prolonged exposure to light and do not freeze.

Images

Flow Cytometry: LumiDye™ PE-Cy5-labeled SpyCatcher (rp218684)

LumiDye™ PE-Cy5-labeled SpyCatcher (rp218684) - Flow Cytometry

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: LumiDye™ PE-Cy5-labeled SpyCatcher (rp218684)

LumiDye™ PE-Cy5-labeled SpyCatcher (rp218684) - Flow Cytometry

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.