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AAT Bioquest

ReadiUse™ Preactivated APC-Cy5.5 Tandem

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.

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. 

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
ReadiUse™ Preactivated PE-Cy5.5 Tandem5656711960000
ReadiUse™ Preactivated PerCP-Cy5.5 Tandem482695350000
ReadiUse™ Preactivated APC-Cy5.5 Maleimide651700700000

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
Page updated on October 4, 2024

Ordering information

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Catalog Number2586
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Physical properties

Solvent

Water

Spectral properties

Extinction coefficient (cm -1 M -1)

700000

Excitation (nm)

651

Emission (nm)

700

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Refrigerated (2-8 °C); Minimize light exposure
UNSPSC12171501

Components

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.
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.