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ReadiUse™ Preactivated PerCP-Cy5.5 Tandem
PerCP (Peridinin-chlorophyll-protein complex) is isolated from Dinophyceae sp. It has an extremely high extinction coefficient, a high quantum efficiency and a large Stokes shift. It is well excited with the Argon laser at 488 nm with its maximum emission peak at 677 nm. PerCP protein is commonly used for fluorescent immunolabeling, particularly in applications involving fluorescent-activated cell sorting (FACS). Its tandem conjugates (such as PerCP-Cy5.5) can be excited with a standard 488 nm laser and emits in the far red at a longer wavelength for multicolor flow cytometric analysis of cells. These multiple emission wavelengths make PerCP- Cyanine conjugates potentially useful fluorochromes for multicolor analysis with FITC, PE and other fluorochromes. PerCP tandem structure may make it more photostable than PerCP alone, which generally photobleaches rapidly with more powerful water-cooled gas lasers. AAT Bioquest offers this preactivated PerCP-Cy5.5 Tandem to facilitate the PerCP-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated PerCP-Cy5.5 tandem is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated PerCP-Cy5.5 tandem is conjugated to a protein via its amino group that is abundant in proteins while SMCC chemistry targets the thiol group that has to be regenerated by the reduction of antibodies.
Example protocol
AT A GLANCE
Important PerCP-Cy5.5 was premodified with our Buccutite™ FOL. Your antibody (or other proteins) is modified with our Buccutite™ MTA to give MTA-modified protein. The MTA-modified protein readily reacts with FOL-modified PerCP Cy5.5 (provided) to give the desired PerCP-Cy5.5-antibody conjugate.
SAMPLE EXPERIMENTAL PROTOCOL
Preparation of pre-activated Antibody with Buccutite™ MTA
- Reconstitute Buccutite™ MTA in DMSO at ~10 mg/mL.
Note Store unused MTA at -20 °C; it can be used for up to two freeze and thaw cycles. - Prepare target antibody (Ab) in pH = 8.5 - 9.0 buffer at a concentration above 1 mg/ml.
- Add the MTA to Ab solution at the ratio of 8 - 10 µg MTA/100 µg Ab.
- Mix well and react at room temperature for 60 minutes, rotating during the reaction.
- Purify the reaction mixture with a desalting column to remove any unreacted MTA. Exchange the buffer to PBS or another buffer of your choice.
- Collect the MTA-activated Ab. Estimate the concentration by 70% yield of the original starting amount.
Conjugate with Pre-activated PerCP-Cy5.5
- Reconstitute pre-activated PerCP-Cy5.5 in 100 µL ddH2O to 10 mg/mL.
Note Reconstituted pre-activated PerCP-Cy5.5 is not stable and can not be stored for more than one month. - Add pre-activated PerCP-Cy5.5 directly to MTA-activated target Ab solution at the ratio of 75 µg PerCP-Cy5.5/100 µg MTA-activated Ab.
- Rotate the mixture for 1 - 2 hours at room temperature.
- The Ab/PerCP-Cy5.5 conjugates are now ready to use.
Note The antibody conjugate should be stored at >0.5 mg/mL in the presence of a carrier protein (e.g., 0.1% bovine serum albumin) and 0.02-0.05% sodium azide.
Note The Ab/PerCP-Cy5.5 can be stored at 4 °C for two months. - Optional: Ab/PerCP-Cy5.5 can be further purified through size exclusion chromatography to get better performance.
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 Tandem | 651 | 700 | 700000 |
| ReadiUse™ Preactivated PerCP-Cy5.5 Maleimide | 482 | 695 | 350000 |
References
View all 2 references: Citation Explorer
Four color compensation.
Authors: Stewart, C C and Stewart, S J
Journal: Cytometry (1999): 161-75
Authors: Stewart, C C and Stewart, S J
Journal: Cytometry (1999): 161-75
A strategy for multiple immunophenotyping by image cytometry: model studies using latex microbeads labeled with seven streptavidin-bound fluorochromes.
Authors: Gothot, A and Grosdent, J C and Paulus, J M
Journal: Cytometry (1996): 214-25
Authors: Gothot, A and Grosdent, J C and Paulus, J M
Journal: Cytometry (1996): 214-25
Page updated on December 17, 2024
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