ReadiUse™ Preactivated APC-Cy5.5 Maleimide

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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

Molecular weight	N/A
Solvent	Water

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	700000
Excitation (nm)	651
Emission (nm)	700

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

Overview SDS Protocol

Example protocol

SAMPLE EXPERIMENTAL PROTOCOL

Reduction of Antibody

Prepare a fresh solution of 1.0 M DTT (15.4 mg/100 µL) in distilled water. Antibody solutions should be at 2 mg/mL or higher for best results. The reduction can be carried out in different buffers for example: MES, phosphate, and TRIS buffers (pH range 6 to 8). The antibody should be concentrated if less than 2 mg/mL.
Add 2 µL of 1.0 M DTT stock per 100 µL of antibody solution and mix well. Let the antibody solution stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
Purify the reduced antibody over a desalting column pre-equilibrated with 50 mM MES Buffer (pH=6.0-6.5) with 2 mM EDTA. (Desalting column: https://www.aatbio.com/products/readiuse-bio-gel-p-6-spin-column?unit=60500)
Measure the Antibody concentration with Nanodrop. (Con. (mg/ml)= A280nm/1.4) Note: The reduced antibody is not stable; the conjugation reaction needs to be carried out the conjugation as soon as possible after purification.

Conjugate with ReadiUse™ Preactivated APC-Cy5.5 Maleimide

Reconstitute ReadiUse™ Preactivated APC-Cy5.5 Maleimide in 100 µL ddH₂O to 10 mg/mL. Note: Reconstituted ReadiUse™ Preactivated APC-Cy5.5 Maleimide are stable at 4 °C for one week, please kept it from light.
Add reduced antibody to pre-activated APC-Cy5.5 directly to at the ratio of 130 µg APC-Cy5.5 /100 µg reduced antibody.
Rotate the mixture for 60-120 mins at room temperature.
After 60 minutes, block the free sulfhydryls on the antibody.
Prepare a fresh solution of 10 mg/mL NEM in DMSO; add 3.4 µL per mg of antibody and rotate for 20 minutes at room temperature.

Purification

Antibody/APC-Cy5.5 conjugate could be further purified through size exclusion chromatography to get best performance. Note: The Antibody/APC-Cy5.5 conjugate solution is recommended to be stored at 2~8 °C and kept from light.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	700000
Excitation (nm)	651
Emission (nm)	700

Product Family

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

References

View all 2 references: Citation Explorer

PE-Cy5.5 conjugates bind to the cells expressing mouse DEC205/CD205.
Authors: Park, Chae Gyu and Rodriguez, Anthony and Steinman, Ralph M
Journal: Journal of immunological methods (2012): 184-90

Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.
Authors: Mittag, Anja and Lenz, Dominik and Gerstner, Andreas O H and Sack, Ulrich and Steinbrecher, Michael and Koksch, Mario and Raffael, Alexander and Bocsi, Jozsef and Tárnok, Attila
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2005): 103-15