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ReadiUse™ Preactivated APC-Cy5.5 Tandem

Our preactivated APC-Cy5.5 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-Cy5.5 (provided) to give the desired APC-Cy5.5-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated APC-Cy5.5 reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
Our preactivated APC-Cy5.5 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-Cy5.5 (provided) to give the desired APC-Cy5.5-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated APC-Cy5.5 reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
Ordering information
Price ()
Catalog Number2586
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)656
Emission (nm)700
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
105000
Extinction coefficient (cm -1 M -1)
700000
Excitation (nm)
656
Emission (nm)
700
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. APC-Cy5.5 is a popular color used in flow cytometry. Its primary absorption peak is at 651 nm with emission peak at~700 nm. AAT Bioquest offers this preactivated APC-Cy5.5 to facilitate the APC-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated APC-Cy5.5 tandem is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated APC-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 APC-Cy5.5 Tandem1 vial (1 mg)
B: Buccutite™ MTA1 vial (100 µg)
C: Spin Desalting ColumnNot 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-Cy5.5
  1. Reconstitute pre-activated APC-Cy5.5 in 100 µL ddH2O to 10 mg/mL.
    Note     Reconstituted pre-activated APC-Cy5.5 could be stored at 4 °C for one month, kept from light.
  2. Add APC-Cy5.5 directly to MTA-activated target Ab solution at the ratio of 130 µg APC-Cy5.5/100 µg MTA-activated Ab.
  3. Rotate the mixture for 60 minutes at room temperature.
  4. The Ab/APC-Cy5.5 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-Cy5.5 conjugates solution could be stored at 4 °C for up to two months, and kept from light.
  5. (Optional) Ab/APC-Cy5.5 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-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 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)656
Emission (nm)700

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
ReadiUse™ Preactivated PE-Cy5.5 Tandem5666711960000
ReadiUse™ Preactivated PerCP-Cy5.5 Tandem489679350000
ReadiUse™ Preactivated APC-Cy5.5 Maleimide656700700000

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