Calculator
ReadiUse™ Preactivated PE-Cy5.5 Maleimide
PE-Cy5.5 is a popular tandem color used in flow cytometry. Its primary absorption peak is at 565 nm with emission peak at ~700 nm. AAT Bioquest offers this preactivated PE-Cy5.5 maleimide to facilitate the PE-Cy5.5 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-Cy5.5 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-Cy5.5 Maleimide
- Reconstitute ReadiUse™ Preactivated PE-Cy5.5 Maleimide in 100 µL ddH2O to 10 mg/mL. Note: Reconstituted ReadiUse™ Preactivated PE-Cy5.5 Maleimide are stable at 4 °C for one week, please kept it from light.
- Add reduced antibody to pre-activated PE-Cy5.5 directly to at the ratio of 250-300 µg PE-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/PE-Cy5.5 conjugate could be further purified through size exclusion chromatography to get best performance. Note: The Antibody/PE-Cy5.5 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-Cy5.5 Tandem | 565 | 671 | 1960000 |
| ReadiUse™ Preactivated APC-Cy5.5 Maleimide | 651 | 700 | 700000 |
| ReadiUse™ Preactivated PerCP-Cy5.5 Maleimide | 482 | 695 | 350000 |
References
View all 5 references: Citation Explorer
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
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
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
Simultaneous flow cytometric measurement of antigen attachment to phagocytes and phagocytosis.
Authors: Laopajon, Witida and Takheaw, Nuchjira and Kasinrerk, Watchara and Pata, Supansa
Journal: Journal of immunoassay & immunochemistry (2016): 527-39
Authors: Laopajon, Witida and Takheaw, Nuchjira and Kasinrerk, Watchara and Pata, Supansa
Journal: Journal of immunoassay & immunochemistry (2016): 527-39
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
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
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
Page updated on December 3, 2024
Safety data sheet