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ReadiUse™ Preactivated PE-Cy5.5 Tandem

Our preactivated PE-Cy5.5 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-Cy5.5 Tandem (provided) to give the desired PE-Cy5.5 Tandem-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated PE-Cy5.5 Tandem reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
Our preactivated PE-Cy5.5 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-Cy5.5 Tandem (provided) to give the desired PE-Cy5.5 Tandem-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated PE-Cy5.5 Tandem reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
Ordering information
Price ()
Catalog Number2581
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight240000
Spectral properties
Extinction coefficient (cm -1 M -1)1960000
Excitation (nm)566
Emission (nm)671
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501
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Show More (64)

OverviewpdfSDSpdfProtocol


Molecular weight
240000
Extinction coefficient (cm -1 M -1)
1960000
Excitation (nm)
566
Emission (nm)
671
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.

Components


A: ReadiUse™ Preactivated PE-Cy5.5 Tandem1 vial (1 mg)
B: Buccutite™ MTA1 vial (100 µg)
C: Spin Desalting ColumnNot Included

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
  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-Cy5.5 Tandem
  1. 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.
  2. 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.
  3. Rotate the mixture for 1 - 2 hours at room temperature.
  4. 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.
  5. Optional: Ab/PE-Cy5.5 Tandem can be further purified through size exclusion chromatography to get better performance. 

Calculators


Common stock solution preparation

Table 1. Volume of appropriate solvent needed to reconstitute specific mass of ReadiUse™ Preactivated PE-Cy5.5 Tandem to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM416.667 nL2.083 µL4.167 µL20.833 µL41.667 µL
5 mM83.333 nL416.667 nL833.333 nL4.167 µL8.333 µL
10 mM41.667 nL208.333 nL416.667 nL2.083 µL4.167 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)1960000
Excitation (nm)566
Emission (nm)671

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
ReadiUse™ Preactivated APC-Cy5.5 Tandem656700700000
ReadiUse™ Preactivated PerCP-Cy5.5 Tandem489679350000
ReadiUse™ Preactivated PE-Cy5.5 Maleimide5666711960000

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