ReadiUse™ Preactivated PE-Cy5 Maleimide

Flow cytometry analysis of whole blood stained with PE-Cy5 anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the PE-Cy5 specific B8-A channel.

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
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)	1960000
Excitation (nm)	565
Emission (nm)	666

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

Molecular weight

N/A

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

1960000

Excitation (nm)

565

Emission (nm)

666

PE-Cy5 is a popular tandem color used in flow cytometry. Its primary absorption peak is at 565 nm with emission peak at ~660 nm. AAT Bioquest offers this preactivated PE-Cy5 maleimide to facilitate the PE-Cy5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. It is prepared from the commonly used crosslinker SMCC. Our preactivated PE-Cy5 maleimide is ready to conjugate the reduced antibodies and other thiol-containing biomolecules.

Example protocol

SAMPLE EXPERIMENTAL PROTOCOL

Reduction of IgG

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 PE-Cy5 Maleimide

Reconstitute ReadiUse™ Preactivated PE-Cy5 Maleimide in 100 µL ddH₂O to 10 mg/mL. Note: Reconstituted ReadiUse™ Preactivated PE-Cy5 Maleimide are stable at 4 °C for one week, please kept it from light.
Add reduced antibody to pre-activated PE-Cy5 directly to at the ratio of 250-300 µg PE-Cy5 /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/PE-Cy5 conjugate could be further purified through size exclusion chromatography to get best performance. Note: The Antibody/PE-Cy5 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)	1960000
Excitation (nm)	565
Emission (nm)	666

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)
ReadiUse™ Preactivated PE-Cy5 Tandem	565	666	1960000
ReadiUse™ Preactivated PE-Cy7 Maleimide	565	778	1960000

Images

Figure 1. Flow cytometry analysis of whole blood stained with PE-Cy5 anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the PE-Cy5 specific B8-A channel.

References

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Journal: Cytometry. Part B, Clinical cytometry (2018): 707-713

Association of Regulatory T Cells with Diabetes Type-1 and Its Renal and Vascular Complications Based on the Expression of Forkhead Box Protein P3 (FoxP3), Helios and Neurophilin-1.
Authors: Khamechian, Tahereh and Irandoust, Behnaz and Mohammadi, Hanieh and Nikoueinejad, Hassan and Akbari, Hossein
Journal: Iranian journal of allergy, asthma, and immunology (2018): 151-157

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Authors: Tian, Lu and Zhang, Lunjia and Gao, Meng and Deng, Zhongshan and Gui, Lin
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Authors: Correia, R P and Rajab, A and Bento, L C and Alexandre, A M and Vaz, A C and Schimidell, D and Pedro, E C and Perin, F S and Nozawa, S T and Barroso, R S and Bacal, N S
Journal: International journal of laboratory hematology (2018): 136-143

CD4+ T cells and natural killer cells: Biomarkers for hepatic fibrosis in human immunodeficiency virus/hepatitis C virus-coinfected patients.
Authors: Laufer, Natalia and Ojeda, Diego and Polo, María Laura and Martinez, Ana and Pérez, Héctor and Turk, Gabriela and Cahn, Pedro and Zwirner, Norberto Walter and Quarleri, Jorge
Journal: World journal of hepatology (2017): 1073-1080

Multiplexed Lipid Bilayers on Silica Microspheres for Analytical Screening Applications.
Authors: Fernandez Oropeza, Nadiezda and Zurek, Nesia A and Galvan-De La Cruz, Mirella and Fabry-Wood, Aurora and Fetzer, Jennifer M and Graves, Steven W and Shreve, Andrew P
Journal: Analytical chemistry (2017): 6440-6447