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ReadiUse™ Preactivated PE-Cy7 Maleimide
PE-Cy7 is a popular tandem color used in flow cytometry. Its primary absorption peak is at 565 nm with emission peak at ~780 nm. AAT Bioquest offers this preactivated PE-Cy7 maleimide to facilitate the PE-Cy7 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-Cy7 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-Cy7 Maleimide
- Reconstitute ReadiUse™ Preactivated PE-Cy7 Maleimide in 100 µL ddH2O to 10 mg/mL. Note: Reconstituted ReadiUse™ Preactivated PE-Cy7 Maleimide are stable at 4 °C for one week, please kept it from light.
- Add reduced antibody to pre-activated PE-Cy7 directly to at the ratio of 250-300 µg PE-Cy7 /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-Cy7 conjugate could be further purified through size exclusion chromatography to get best performance. Note: The Antibody/PE-Cy7 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-Cy7 Tandem | 565 | 778 | 1960000 |
| ReadiUse™ Preactivated PE-Cy5 Maleimide | 565 | 666 | 1960000 |
| ReadiUse™ Preactivated APC-Cy7 Maleimide | 651 | 779 | 700000 |
| ReadiUse™ Preactivated PerCP-Cy7 Maleimide | 757 | 783 | 350000 |
References
View all 37 references: Citation Explorer
Recombinant thrombomodulin attenuates hyper-inflammation and glycocalyx damage in a murine model of Streptococcus pneumoniae-induced sepsis.
Authors: Watanabe, Eizo and Akamatsu, Toshinobu and Ohmori, Masaaki and Kato, Mayu and Takeuchi, Noriko and Ishiwada, Naruhiko and Nishimura, Rintaro and Hishiki, Haruka and Fujimura, Lisa and Ito, Chizuru and Hatano, Masahiko
Journal: Cytokine (2022): 155723
Authors: Watanabe, Eizo and Akamatsu, Toshinobu and Ohmori, Masaaki and Kato, Mayu and Takeuchi, Noriko and Ishiwada, Naruhiko and Nishimura, Rintaro and Hishiki, Haruka and Fujimura, Lisa and Ito, Chizuru and Hatano, Masahiko
Journal: Cytokine (2022): 155723
Cordyceps militaris extract induces apoptosis and pyroptosis via caspase-3/PARP/GSDME pathways in A549 cell line.
Authors: Hu, Zixuan and Lai, Yijing and Ma, Chaoya and Zuo, Lina and Xiao, Guanlin and Gao, Haili and Xie, Biyuan and Huang, Xuejun and Gan, Haining and Huang, Dane and Yao, Nan and Feng, Baoguo and Ru, JieXia and Chen, Yuxing and Cai, Dake
Journal: Food science & nutrition (2022): 21-38
Authors: Hu, Zixuan and Lai, Yijing and Ma, Chaoya and Zuo, Lina and Xiao, Guanlin and Gao, Haili and Xie, Biyuan and Huang, Xuejun and Gan, Haining and Huang, Dane and Yao, Nan and Feng, Baoguo and Ru, JieXia and Chen, Yuxing and Cai, Dake
Journal: Food science & nutrition (2022): 21-38
Development and validation of a sensitive flow cytometric method for determining CECs in RBC products.
Authors: Li, Wenhui and Acker, Jason P
Journal: Clinica chimica acta; international journal of clinical chemistry (2022): 119-125
Authors: Li, Wenhui and Acker, Jason P
Journal: Clinica chimica acta; international journal of clinical chemistry (2022): 119-125
Routine flow cytometry approach for the evaluation of solid tumor neoplasms and immune cells in minimally invasive samples.
Authors: Quirós-Caso, Covadonga and Arias Fernández, Tamara and Fonseca-Mourelle, Ariana and Torres, Héctor and Fernández, Luis and Moreno-Rodríguez, Maria and Ariza-Prota, Miguel Ángel and López-González, Francisco Julián and Carvajal-Álvarez, Miguel and Alonso-Álvarez, Sara and Moro-García, Marco Antonio and Colado, Enrique
Journal: Cytometry. Part B, Clinical cytometry (2022)
Authors: Quirós-Caso, Covadonga and Arias Fernández, Tamara and Fonseca-Mourelle, Ariana and Torres, Héctor and Fernández, Luis and Moreno-Rodríguez, Maria and Ariza-Prota, Miguel Ángel and López-González, Francisco Julián and Carvajal-Álvarez, Miguel and Alonso-Álvarez, Sara and Moro-García, Marco Antonio and Colado, Enrique
Journal: Cytometry. Part B, Clinical cytometry (2022)
The ISCCA flow protocol for the monitoring of anti-CD20 therapies in autoimmune disorders.
Authors: Gatti, Arianna and Buccisano, Francesco and Scupoli, Maria T and Brando, Bruno
Journal: Cytometry. Part B, Clinical cytometry (2021): 194-205
Authors: Gatti, Arianna and Buccisano, Francesco and Scupoli, Maria T and Brando, Bruno
Journal: Cytometry. Part B, Clinical cytometry (2021): 194-205
Page updated on November 21, 2024
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