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ReadiUse™ Preactivated PE-Cy5.5 Tandem
PE-Cy5.5 is a popular color used in flow cytometry. Its primary absorption peak is at 565 nm with emission peak at~700 nm. The filter sets of 682/33 nm and 695/40 nm are recommended for this tandem color. AAT Bioquest offers this preactivated PE-Cy5.5 to facilitate the PE-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated PE-Cy5.5 tandem is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated PE-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 PE-Cy5.5 Tandem 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 PE-Cy5.5 Tandem (provided) to give the desired PE-Cy5.5 Tandem-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 PE-Cy5.5 Tandem
- Reconstitute pre-activated PE-Cy5.5 Tandem in 100 µL ddH2O to 10 mg/mL.
Note Reconstituted pre-activated PE-Cy5.5 Tandem is not stable and can not be stored for more than one month. - Add pre-activated PE-Cy5.5 Tandem directly to MTA-activated target Ab solution at the ratio of 300 µg PE-Cy5.5 Tandem/100 µg MTA-activated Ab.
- Rotate the mixture for 1 - 2 hours at room temperature.
- The Ab/PE-Cy5.5 Tandem 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/PE-Cy5.5 Tandem can be stored at 4 °C for two months. - Optional: Ab/PE-Cy5.5 Tandem 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 APC-Cy5.5 Tandem | 651 | 700 | 700000 |
| ReadiUse™ Preactivated PerCP-Cy5.5 Tandem | 482 | 695 | 350000 |
| ReadiUse™ Preactivated PE-Cy5.5 Maleimide | 565 | 671 | 1960000 |
References
View all 46 references: Citation Explorer
Chromophore attachment to phycobiliprotein beta-subunits: phycocyanobilin:cysteine-beta84 phycobiliprotein lyase activity of CpeS-like protein from Anabaena Sp. PCC7120
Authors: Zhao KH, Su P, Li J, Tu JM, Zhou M, Bubenzer C, Scheer H.
Journal: J Biol Chem (2006): 8573
Authors: Zhao KH, Su P, Li J, Tu JM, Zhou M, Bubenzer C, Scheer H.
Journal: J Biol Chem (2006): 8573
Excitation energy transfer from phycobiliprotein to chlorophyll d in intact cells of Acaryochloris marina studied by time- and wavelength-resolved fluorescence spectroscopy
Authors: Petrasek Z, Schmitt FJ, Theiss C, Huyer J, Chen M, Larkum A, Eichler HJ, Kemnitz K, Eckert HJ.
Journal: Photochem Photobiol Sci (2005): 1016
Authors: Petrasek Z, Schmitt FJ, Theiss C, Huyer J, Chen M, Larkum A, Eichler HJ, Kemnitz K, Eckert HJ.
Journal: Photochem Photobiol Sci (2005): 1016
Single-molecule spectroscopy selectively probes donor and acceptor chromophores in the phycobiliprotein allophycocyanin
Authors: Loos D, Cotlet M, De Schryver F, Habuchi S, Hofkens J.
Journal: Biophys J (2004): 2598
Authors: Loos D, Cotlet M, De Schryver F, Habuchi S, Hofkens J.
Journal: Biophys J (2004): 2598
Isolation and characterisation of phycobiliprotein rich mutant of cyanobacterium Synechocystis sp
Authors: Prasanna R, Dhar DW, Dominic TK, Tiwari ON, Singh PK.
Journal: Acta Biol Hung (2003): 113
Authors: Prasanna R, Dhar DW, Dominic TK, Tiwari ON, Singh PK.
Journal: Acta Biol Hung (2003): 113
Evaluation of Tolypothrix germplasm for phycobiliprotein content
Authors: Prasanna R, Prasanna BM, Mohammadi SA, Singh PK.
Journal: Folia Microbiol (Praha) (2003): 59
Authors: Prasanna R, Prasanna BM, Mohammadi SA, Singh PK.
Journal: Folia Microbiol (Praha) (2003): 59
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