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ReadiUse™ Preactivated APC-iFluor® 750 Tandem

Flow cytometry analysis of whole blood cells stained with APC/iFluor® 750 anti-human CD4 antibody (Clone: SK3). The fluorescence signal was monitored using an Aurora flow cytometer in the APC/iFluor® 750 specific R7-A channel.
Flow cytometry analysis of whole blood cells stained with APC/iFluor® 750 anti-human CD4 antibody (Clone: SK3). The fluorescence signal was monitored using an Aurora flow cytometer in the APC/iFluor® 750 specific R7-A channel.
Spectrum for APC-iFluor 750
Ordering information
Price ()
Catalog Number2571
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 weight105000
Spectral properties
Extinction coefficient (cm -1 M -1)700000
Excitation (nm)651
Emission (nm)791
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Molecular weight
105000
Extinction coefficient (cm -1 M -1)
700000
Excitation (nm)
651
Emission (nm)
791
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. Our APC-iFluor® 750 Tandem is an excellent replacement for APC-Cy7® and APC-Alexa Fluor® 750 Tandem due to their similar spectral properties. Our APC-iFluor® 750 Tandem is more photostable than APC-Cy7® Tandem. ReadiUse™ Preactivated APC-iFluor® 750 Tandem is an activated APC protein, and can be easily conjugated to antibodies with much higher conjugation yield than the conventional APC.

Components


A: ReadiUse™ Preactivated APC-iFluor™ 750 Tandem1 vial (1 mg)
B: Buccutite™ MTA 1 vial (100 µg)
C: Spin Desalting Column Not Included

Example protocol


SAMPLE EXPERIMENTAL PROTOCOL

Preparation of pre-activated 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 1 mg/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 room temperature 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. 

Conjugation with pre-activated APC-iFluor™ 750 Tandem
  1. Reconstitute pre-activated APC-iFluor™ 750 Tandem in 100 µL ddH2O to 10 mg/mL.
    Note     Reconstituted pre-activated APC-iFluor™ 750 Tandem could be stored at 4 °C for one month, kept from light.
  2. Add APC-iFluor™ 750 Tandem directly to MTA-activated target Ab solution at the ratio of 130 µg APC-iFluor™ 750 Tandem/100 µg MTA-activated Ab.
  3. Rotate the mixture for 60 minutes at room temperature.
  4. The Ab/APC-iFluor™ 750 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. The Ab/APC-iFluor™ 750 Tandem conjugates solution could be stored at 4 °C for up to two months, and kept from light.
  5. (Optional) Ab/APC-iFluor™ 750 Tandem conjugates could be further purified through size exclusion chromatography to get best performance. 

Calculators


Common stock solution preparation

Table 1. Volume of appropriate solvent needed to reconstitute specific mass of ReadiUse™ Preactivated APC-iFluor® 750 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 mM952.381 nL4.762 µL9.524 µL47.619 µL95.238 µL
5 mM190.476 nL952.381 nL1.905 µL9.524 µL19.048 µL
10 mM95.238 nL476.19 nL952.381 nL4.762 µL9.524 µL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)700000
Excitation (nm)651
Emission (nm)791

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
APC-iFluor® 750 Tandem754776700000
PE-iFluor® 750 Tandem566778-
APC-iFluor® 700 Tandem685710700000
APC-iFluor® 710 Tandem---
APC-iFluor® 800 Tandem804660700000
APC-iFluor®A7 Tandem---

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