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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.
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.
Ordering information
Price ()
Catalog Number2717
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weightN/A
SolventWater
Spectral properties
Extinction coefficient (cm -1 M -1)1960000
Excitation (nm)565
Emission (nm)666
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageRefrigerated (2-8 °C); Minimize light exposure
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OverviewpdfSDSpdfProtocol


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
  1. 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.
  2. 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).
  3. 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)
  4. 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
  1. 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.
  2. Add reduced antibody to pre-activated PE-Cy5 directly to at the ratio of 250-300 µg PE-Cy5 /100 µg reduced antibody.
  3. Rotate the mixture for 60-120 mins at room temperature.
  4. After 60 minutes, block the free sulfhydryls on the antibody.
  5. 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
  1. 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
spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)1960000
Excitation (nm)565
Emission (nm)666

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
ReadiUse™ Preactivated PE-Cy5 Tandem5656661960000
ReadiUse™ Preactivated PE-Cy7 Maleimide5667781960000

References


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Indicator of Inflammation and NETosis-Low-Density Granulocytes as a Biomarker of Autoimmune Hepatitis.
Authors: Domerecka, Weronika and Homa-Mlak, Iwona and Mlak, Radosław and Michalak, Agata and Wilińska, Agnieszka and Kowalska-Kępczyńska, Anna and Dreher, Piotr and Cichoż-Lach, Halina and Małecka-Massalska, Teresa
Journal: Journal of clinical medicine (2022)
Expression of CD163 and HLA-DR molecules on the monocytes in chronic lymphocytic leukemia patients.
Authors: Kowalska, Wioleta
Journal: Folia histochemica et cytobiologica (2020): 17-24
Anti-epithelial cell adhesion molecule RNA aptamer-conjugated liposomal doxorubicin as an efficient targeted therapy in mice bearing colon carcinoma tumor model.
Authors: Mashreghi, Mohammad and Zamani, Parvin and Karimi, Maryam and Mehrabian, Amin and Arabsalmani, Mahdieh and Zarqi, Javad and Moosavian, Seyedeh Alia and Jaafari, Mahmoud Reza
Journal: Biotechnology progress (2020): e3116
Image cytometry as an alternative to flow cytometry for the transplant histocompatibility crossmatch assay.
Authors: Ramon, Daniel S and Franks, Thomas and Jaramillo, Andrés and Paradis, Benjamin D and Chan, Leo Li-Ying
Journal: HLA (2019): 436-444
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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
A Handy Liquid Metal Based Non-Invasive Electrophoretic Particle Microtrap.
Authors: Tian, Lu and Zhang, Lunjia and Gao, Meng and Deng, Zhongshan and Gui, Lin
Journal: Micromachines (2018)
A ten-color tube with dried antibody reagents for the screening of hematological malignancies.
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