ReadiUse™ Preactivated PE-iFluor™ 647 Tandem

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<p>Our preactivated PE 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 (provided) to give the desired PE-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated PE reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.</p>
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Unit Size: Cat No: Price (USD): Qty:
1 mg 2577 $345


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)568/666
SolventWater
Storage Refrigerated (2-4 °C)
Minimize light exposure
Category Protein-Based Tags
Phycobiliproteins
Related Protein Dyes
R-Phycoerythrin (PE) is isolated from red algae. Its primary absorption peak is at 565 nm with secondary peaks at 496 and 545 nm. Our RPE-iFluor™ 647 Tandem is an excellent replacement for RPE-Cy5® and RPE-Alexa Fluor® 647 Tandem since they have almost identical spectra. ReadiUse™ Preactivated PE-iFluor™ 647 Tandem is an activated PE protein, and can be easily conjugated to antibodies with much higher conjugation yield than the conventional PE.




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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
  1. Pre-activate Antibody with MTA
    1. Reconstitute MTA in DMSO at ~10 mg/mL.
      Note: Please store unused MTA at -20°C and could be used up to two freeze and thaw cycles.
    2. Add MTA to Ab solution at the ratio of 8~10ug MTA /100 ug Ab.
    3. Mix well and react at RT for 60 minutes, rotating during the reaction
    4. Purify the reaction mixture with desalting column to remove unreacted MTA and exchange buffer to PBS or buffer of your choice.
    5. Collect the MTA-activated Ab, and estimate the concentration by 70% yield of the original starting amount.

  2. Conjugate with Pre-activated PE-iFluor647 Tandem
    1. Reconstitute pre-activated PE- iFluor647 Tandem in 100 µL ddH2O to 10mg/mL.
      Note: Reconstituted pre-activated PE- iFluor647 Tandem is not stable and could be stored at 4°C for one month, please kept from light.
    2. Add PE-iFluor647 directly to MTA-activated target Ab solution (from 1.5) at the ratio of 300µg PE-iFluor647 /100µg MTA-activated Ab.
    3. Rotate the mixture for 1~2 hours at room temperature
    4. The Ab/ PE-iFluor647 conjugate is now ready to use.
      Optional: Ab/ PE-iFluor647 Conjugate could be further purified through size exclusion chromatography to get best performance.
      Note1: 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.
      Note2: The Ab/ PE-iFluor647 conjugate solution could be stored at 4 °C for two months and kept from light.





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References

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

Evaluation of Tolypothrix germplasm for phycobiliprotein content
Authors: Prasanna R, Prasanna BM, Mohammadi SA, Singh PK.
Journal: Folia Microbiol (Praha) (2003): 59

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

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, Tandeau 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

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-Fab conjugates as probes for single particle fluorescence imaging
Authors: Triantafilou K, Triantafilou M, Wilson KM.
Journal: Cytometry (2000): 226

[Phycobiliprotein and fluorescence immunological assay]
Authors: Wu P.
Journal: Sheng Li Ke Xue Jin Zhan (2000): 82


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