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ReadiUse™ Preactivated PE-Cy5 Maleimide
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
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 |
References
View all 50 references: Citation Explorer
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)
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
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
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
Authors: Ramon, Daniel S and Franks, Thomas and Jaramillo, Andrés and Paradis, Benjamin D and Chan, Leo Li-Ying
Journal: HLA (2019): 436-444
Initial flow cytometric evaluation of the Clearllab lymphoid screen.
Authors: Hedley, B D and Cheng, G and Luider, J and Kern, W and Lozanski, G and Chin-Yee, I and Lowes, L E and Keeney, M and Careaga, D and Magari, R and Tejidor, L
Journal: Cytometry. Part B, Clinical cytometry (2018): 707-713
Authors: Hedley, B D and Cheng, G and Luider, J and Kern, W and Lozanski, G and Chin-Yee, I and Lowes, L E and Keeney, M and Careaga, D and Magari, R and Tejidor, L
Journal: Cytometry. Part B, Clinical cytometry (2018): 707-713
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