ReadiUse™ Preactivated PerCP-Cy5.5 Tandem

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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Shipping	Standard overnight for United States, inquire for international

Physical properties

Solvent

Water

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	350000
Excitation (nm)	482
Emission (nm)	695

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Refrigerated (2-8 °C); Minimize light exposure
UNSPSC	12171501

Overview SDS Protocol

Extinction coefficient (cm ^-1 M ^-1)

350000

Excitation (nm)

482

Emission (nm)

695

PerCP (Peridinin-chlorophyll-protein complex) is isolated from Dinophyceae sp. It has an extremely high extinction coefficient, a high quantum efficiency and a large Stokes shift. It is well excited with the Argon laser at 488 nm with its maximum emission peak at 677 nm. PerCP protein is commonly used for fluorescent immunolabeling, particularly in applications involving fluorescent-activated cell sorting (FACS). Its tandem conjugates (such as PerCP-Cy5.5) can be excited with a standard 488 nm laser and emits in the far red at a longer wavelength for multicolor flow cytometric analysis of cells. These multiple emission wavelengths make PerCP- Cyanine conjugates potentially useful fluorochromes for multicolor analysis with FITC, PE and other fluorochromes. PerCP tandem structure may make it more photostable than PerCP alone, which generally photobleaches rapidly with more powerful water-cooled gas lasers. AAT Bioquest offers this preactivated PerCP-Cy5.5 Tandem to facilitate the PerCP-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated PerCP-Cy5.5 tandem is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated PerCP-Cy5.5 tandem is conjugated to a protein via its amino group that is abundant in proteins while SMCC chemistry targets the thiol group that has to be regenerated by the reduction of antibodies.

Components

Example protocol

AT A GLANCE

Important PerCP-Cy5.5 was premodified with our Buccutite™ FOL. Your antibody (or other proteins) is modified with our Buccutite™ MTA to give MTA-modified protein. The MTA-modified protein readily reacts with FOL-modified PerCP Cy5.5 (provided) to give the desired PerCP-Cy5.5-antibody conjugate.

SAMPLE EXPERIMENTAL PROTOCOL

Preparation of pre-activated Antibody with Buccutite™ MTA

Reconstitute Buccutite™ MTA in DMSO at ~10 mg/mL.
Note Store unused MTA at -20 °C; it can be used for up to two freeze and thaw cycles.
Prepare target antibody (Ab) in pH = 8.5 - 9.0 buffer at a concentration above 1 mg/ml.
Add the MTA to Ab solution at the ratio of 8 - 10 µg MTA/100 µg Ab.
Mix well and react at room temperature for 60 minutes, rotating during the reaction.
Purify the reaction mixture with a desalting column to remove any unreacted MTA. Exchange the buffer to PBS or another buffer of your choice.
Collect the MTA-activated Ab. Estimate the concentration by 70% yield of the original starting amount.

Conjugate with Pre-activated PerCP-Cy5.5

Reconstitute pre-activated PerCP-Cy5.5 in 100 µL ddH₂O to 10 mg/mL.
Note Reconstituted pre-activated PerCP-Cy5.5 is not stable and can not be stored for more than one month.
Add pre-activated PerCP-Cy5.5 directly to MTA-activated target Ab solution at the ratio of 75 µg PerCP-Cy5.5/100 µg MTA-activated Ab.
Rotate the mixture for 1 - 2 hours at room temperature.
The Ab/PerCP-Cy5.5 conjugates are now ready to use.
Note The antibody conjugate should be stored at >0.5 mg/mL in the presence of a carrier protein (e.g., 0.1% bovine serum albumin) and 0.02-0.05% sodium azide.
Note The Ab/PerCP-Cy5.5 can be stored at 4 °C for two months.
Optional: Ab/PerCP-Cy5.5 can be further purified through size exclusion chromatography to get better performance.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	350000
Excitation (nm)	482
Emission (nm)	695

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)
ReadiUse™ Preactivated PE-Cy5.5 Tandem	565	671	1960000
ReadiUse™ Preactivated APC-Cy5.5 Tandem	651	700	700000
ReadiUse™ Preactivated PerCP-Cy5.5 Maleimide	482	695	350000

Images

Figure 1. Conjugation scheme for ReadiUse™ Preactivated PerCP-Cy5.5 Tandem. The activated PerCP-Cy5.5 tandem is premodified with our Buccutite™ FOL and can be readily used for conjugation. To conjugate your desired antibody or protein, first modify it with our Buccutite™ MTA (provided) and then mix both components. The Buccutite™ MTA-modified protein will readily react with the Buccutite™ FOL-activated PerCP-Cy5.5 tandem to give the desired PerCP-Cy5.5-antibody conjugate in much higher yield than SMCC chemistry. In addition our preactivated PerCP-Cy5.5 tandem reacts with MTA-modified biopolymers at a much lower concentration than SMCC chemistry.

References

View all 2 references: Citation Explorer

Four color compensation.
Authors: Stewart, C C and Stewart, S J
Journal: Cytometry (1999): 161-75

A strategy for multiple immunophenotyping by image cytometry: model studies using latex microbeads labeled with seven streptavidin-bound fluorochromes.
Authors: Gothot, A and Grosdent, J C and Paulus, J M
Journal: Cytometry (1996): 214-25