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AAT Bioquest

ReadiUse™ Preactivated PE-Cy5 Tandem

Our preactivated PE-Cy5 Tandem was premodified with our Buccutite™ FOL (provided). Your antibody (or other proteins) is modified with our Buccutite™ MTA (provided as free sample) to give MTA-modified protein (such as antibody). The MTA-modified protein readily reacts with FOL-modified PE-Cy5 Tandem (provided) to give the desired PE-Cy5 Tandem-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated PE-Cy5 Tandem reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
Our preactivated PE-Cy5 Tandem was premodified with our Buccutite™ FOL (provided). Your antibody (or other proteins) is modified with our Buccutite™ MTA (provided as free sample) to give MTA-modified protein (such as antibody). The MTA-modified protein readily reacts with FOL-modified PE-Cy5 Tandem (provided) to give the desired PE-Cy5 Tandem-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated PE-Cy5 Tandem reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
Our preactivated PE-Cy5 Tandem was premodified with our Buccutite™ FOL (provided). Your antibody (or other proteins) is modified with our Buccutite™ MTA (provided as free sample) to give MTA-modified protein (such as antibody). The MTA-modified protein readily reacts with FOL-modified PE-Cy5 Tandem (provided) to give the desired PE-Cy5 Tandem-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated PE-Cy5 Tandem reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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Physical properties
Molecular weight~240000
SolventWater
Spectral properties
Extinction coefficient (cm -1 M -1)1960000
Excitation (nm)565
Emission (nm)666
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageRefrigerated (2-8 °C); Minimize light exposure
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


Molecular weight
~240000
Extinction coefficient (cm -1 M -1)
1960000
Excitation (nm)
565
Emission (nm)
666
PE-Cy5 is a popular color used in flow cytometry. Its primary absorption peak is at 565 nm with emission peak at 666 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 to facilitate the PE-Cy5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated PE-Cy5 tandem is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated PE-Cy5 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.

Components


Example protocol


AT A GLANCE

Important      PE-Cy5 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 Tandem (provided) to give the desired PE-Cy5 Tandem-antibody conjugate.

SAMPLE EXPERIMENTAL PROTOCOL

Preparation of pre-activated Antibody with Buccutite™ MTA
  1. 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.
  2. Prepare target antibody (Ab) in pH = 8.5 - 9.0 buffer at a concentration above 1 mg/ml.
  3. Add the MTA to Ab solution at the ratio of 8 - 10 µg MTA/100 µg Ab.
  4. Mix well and react at room temperature for 60 minutes, rotating during the reaction.
  5. Purify the reaction mixture with a desalting column to remove any unreacted MTA. Exchange the buffer to PBS or another buffer of your choice.
  6. Collect the MTA-activated Ab. Estimate the concentration by 70% yield of the original starting amount. 

Conjugate with Pre-activated PE-Cy5 Tandem
  1. Reconstitute pre-activated PE-Cy5 Tandem in 100 µL ddH2O to 10 mg/mL.
    Note     Reconstituted pre-activated PE-Cy5 Tandem is not stable and can not be stored for more than one month.
  2. Add pre-activated PE-Cy5 Tandem directly to MTA-activated target Ab solution at the ratio of 300 µg PE-Cy5 Tandem/100 µg MTA-activated Ab.
  3. Rotate the mixture for 1 - 2 hours at room temperature.
  4. The Ab/PE-Cy5 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 Tandem can be stored at 4 °C for two months.
  5. Optional: Ab/PE-Cy5 Tandem can be further purified through size exclusion chromatography to get better performance. 

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)1960000
Excitation (nm)565
Emission (nm)666

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
ReadiUse™ Preactivated PE-Cy7 Tandem5657781960000
ReadiUse™ Preactivated PE-Cy5 Maleimide5656661960000

Images


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
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
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
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
Evaluation of Tolypothrix germplasm for phycobiliprotein content
Authors: Prasanna R, Prasanna BM, Mohammadi SA, Singh PK.
Journal: Folia Microbiol (Praha) (2003): 59
Co-ordinated expression of phycobiliprotein operons in the chromatically adapting cyanobacterium Calothrix PCC 7601: a role for RcaD and RcaG
Authors: Noubir S, Luque I, Ochoa de Alda JA, Perewoska I, T and eau de Marsac N, Cobley JG, Houmard J.
Journal: Mol Microbiol (2002): 749
Phycobiliprotein genes of the marine photosynthetic prokaryote Prochlorococcus: evidence for rapid evolution of genetic heterogeneity
Authors: Ting CS, Rocap G, King J, Chisholm SW.
Journal: Microbiology (2001): 3171
Phycobiliprotein-Fab conjugates as probes for single particle fluorescence imaging
Authors: Triantafilou K, Triantafilou M, Wilson KM.
Journal: Cytometry (2000): 226
Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon
Authors: Zhao KH, Deng MG, Zheng M, Zhou M, Parbel A, Storf M, Meyer M, Strohmann B, Scheer H.
Journal: FEBS Lett (2000): 9
Phycobiliprotein and fluorescence immunological assay
Authors: Wu P., undefined
Journal: Sheng Li Ke Xue Jin Zhan (2000): 82