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.
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Freadiuse-preactivated-apc-cy7-tandem%2Ffigure-for-readiuse-preactivated-apc-cy7-tandem_WK0Rz.png&w=640&q=75)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Freadiuse-preactivated-apc-cy7-tandem%2Ffigure-for-readiuse-preactivated-apc-cy7-tandem_WK0Rz.png&w=640&q=75)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Freadiuse-preactivated-apc-cy7-tandem%2Ffigure-for-readiuse-preactivated-apc-cy7-tandem_WK0Rz.png&w=128&q=25)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Freadiuse-preactivated-apc-cy7-tandem%2Ffigure-for-readiuse-preactivated-apc-cy7-tandem_AVGhP.png&w=128&q=25)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Freadiuse-preactivated-apc-cy7-tandem%2Ffigure-for-readiuse-preactivated-apc-cy7-tandem_Iye1P.png&w=128&q=25)
![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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Freadiuse-preactivated-apc-cy7-tandem%2Ffigure-for-readiuse-preactivated-apc-cy7-tandem_RTEKY.jpg&w=128&q=25)
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