ReadiUse™ Preactivated APC-Cy7 Tandem

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	~105000
Solvent	Water

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	700000
Excitation (nm)	651
Emission (nm)	779

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Refrigerated (2-8 °C); Minimize light exposure
UNSPSC	12171501

Direct upgrades

ReadiUse™ Preactivated APC-iFluor® 750 Tandem

Overview SDS Protocol

Upgrade: ReadiUse™ Preactivated APC-iFluor® 750 Tandem

Molecular weight

~105000

Extinction coefficient (cm ^-1 M ^-1)

700000

Excitation (nm)

651

Emission (nm)

779

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-Cy7 is a popular color used in flow cytometry. Its primary absorption peak is at 651 nm with emission peak at~780 nm. AAT Bioquest offers this preactivated APC-Cy7 to facilitate the APC-Cy7 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated APC-Cy7 tandem is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated APC-Cy7 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

Example protocol

SAMPLE EXPERIMENTAL PROTOCOL

Preparation of pre-activated antibody with Buccutite™ MTA

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.
Prepare target antibody (Ab) in pH = 8.5 - 9.0 buffer at concentration above 1 mg/mL.
Add Buccutite™ MTA to Ab solution at the ratio of 8 - 10 µg Buccutite™ MTA/100 µg Ab.
Mix well and react at room temperature for 60 minutes, rotating during the reaction.
Purify the reaction mixture with desalting column to remove unreacted Buccutite™ MTA and exchange buffer to PBS or buffer of your choice.
Collect the Buccutite™ MTA-activated Ab, and estimate the concentration by 70% yield of the original starting amount.

Conjugation with pre-activated APC-Cy7

Reconstitute pre-activated APC-Cy7 in 100 µL ddH₂O to 10 mg/mL.
Note Reconstituted pre-activated APC-Cy7 could be stored at 4 °C for one month, kept from light.
Add APC-Cy7 directly to MTA-activated target Ab solution at the ratio of 130 µg APC-Cy7/100 µg MTA-activated Ab.
Rotate the mixture for 60 minutes at room temperature.
The Ab/APC-Cy7 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-Cy7 conjugates solution could be stored at 4 °C for up to two months, and kept from light.
(Optional) Ab/APC-Cy7 conjugates could be further purified through size exclusion chromatography to get best performance.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	700000
Excitation (nm)	651
Emission (nm)	779

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)
ReadiUse™ Preactivated PE-Cy7 Tandem	565	778	1960000
ReadiUse™ Preactivated APC-Cy7 Maleimide	651	779	700000

Images

Figure 1. Flow cytometry analysis of whole blood stained with APC-Cy7 anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the APC-Cy7 specific R7-A channel.

Figure 2. Flow cytometry analysis of PBMC stained with APC-Cy7 anti-human CD3 *UCHT1* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the APC-Cy7 specific R7-A channel.

Figure 3. Flow cytometry analysis of whole blood stained with APC-Cy7 anti-human CD4 *RPA-T4* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the APC-Cy7 specific R7-A channel.

Figure 4. Our preactivated APC-Cy7 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-Cy7 (provided) to give the desired APC-Cy7-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated APC-Cy7 reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.

Citations

View all 1 citations: Citation Explorer

Rapid clonal identification of biallelic CRISPR/Cas9 knock-ins using SNEAK PEEC
Authors: Singh, Sameer and Banerjee, Anoosha and Vanden Broeck, Arnaud and Klinge, Sebastian
Journal: Scientific Reports (2023): 1719

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