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ReadiUse™ Preactivated APC-Cy7 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-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.
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
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 |
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
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
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
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
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
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
Authors: Prasanna R, Prasanna BM, Mohammadi SA, Singh PK.
Journal: Folia Microbiol (Praha) (2003): 59
Page updated on November 20, 2024
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