ReadiUse™ Preactivated APC-iFluor™ 700 Tandem

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<p>Our preactivated APC 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 APC (provided) to give the desired APC-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated APC 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 2570 $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)651/710
SolventWater
Storage Refrigerated (2-4 °C)
Minimize light exposure
Category Protein-Based Tags
Phycobiliproteins
Related Protein Dyes
Allophycocyanin (APC) is a phycobiliprotein isolated from Spirulina sp., a blue-green alga. Like other phycobiliproteins, APC is fluorescent, with an extremely high absorptivity and a high quantum efficiency. It is a protein which can be easily linked to antibodies and other proteins by conventional protein cross-linking techniques without altering its spectral characteristics. Our APC-iFluor™ 700 Tandem is an excellent replacement for APC-Alexa Fluor® 700 Tandem since they have almost identical spectra. On some antibodies, our APC-iFluor™ 700 Tandem is much brighter than APC-Alexa Fluor® 700 Tandem with a higher stain index. ReadiUse™ Preactivated APC-iFluor™ 700 Tandem is an activated APC protein, and can be easily conjugated to antibodies with much higher conjugation yield than the conventional APC.




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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 Buccutite™ MTA
    1. Reconstitute Buccutite™ MTA in DMSO at ~10 mg/mL.
      Note: Please store unused Buccutite™ MTA at -20°C and could be used up to two freeze and thaw cycles
    2. Prepare target antibody (Ab) in pH = 8.5~9.0 buffer at concentration above 1mg/ml.
    3. Add Buccutite™ MTA to Ab solution at the ratio of 8~10 µg Buccutite™ MTA /100 µg Ab.
    4. Mix well and react at RT for 60 minutes, rotating during the reaction.
    5. Purify the reaction mixture with desalting column to remove unreacted Buccutite™ MTA and exchange buffer to PBS or buffer of your choice.
    6. Collect the Buccutite™ MTA-activated Ab, and estimate the concentration by 70% yield of the original starting amount.

  2. Conjugate with pre-activated APC, APC-ifluors, or APC-ifluor tandems
    1. Reconstitute pre-activated APC, APC, APC-ifluors, or APC-ifluor tandems in 100 µL ddH2O to 10mg/mL.
      Note: Reconstituted pre-activated APC, APC-ifluors, or APC-ifluor tandems are not stable and could be stored at 4 °C for one month, kept from light.
    2. Add APC, APC-ifluors, or APC-ifluor tandems directly to MTA-activated target Ab solution (from 1.5) at the ratio of 130 µg APC, APC-ifluors, or APC-ifluor tandems /100µg MTA-activated Ab.
    3. Rotate the mixture for 1 hour at room temperature
    4. The Ab/APC, APC-ifluors, or APC-ifluor tandems conjugates are now ready to use.
      Note 1(Optional): Ab/APC, APC-ifluors, or APC-ifluor tandems conjugates could be further purified through size exclusion chromatography to get best performance.
      Note 2: 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 3: The Ab/APC, APC-ifluors, or APC-ifluor tandems conjugates solution could be stored at 4 °C for up to 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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