ReadiUse™ Preactivated APC-iFluor® 750 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. Our APC-iFluor® 750 Tandem is an excellent replacement for APC-Cy7® and APC-Alexa Fluor® 750 Tandem due to their similar spectral properties. Our APC-iFluor® 750 Tandem is more photostable than APC-Cy7® Tandem. ReadiUse™ Preactivated APC-iFluor® 750 Tandem is an activated APC protein, and can be easily conjugated to antibodies with much higher conjugation yield than the conventional APC.
![Flow cytometry analysis of whole blood cells stained with APC/iFluor® 750 anti-human CD4 antibody (Clone: SK3). The fluorescence signal was monitored using an Aurora spectral flow cytometer in the APC/iFluor® 750 specific R7-A channel.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Freadiuse-preactivated-apc-ifluor-750-tandem%2Ffigure-for-readiuse-preactivated-apc-ifluor-750-tandem_VNyHD.png&w=640&q=75)
![Flow cytometry analysis of whole blood cells stained with APC/iFluor® 750 anti-human CD4 antibody (Clone: SK3). The fluorescence signal was monitored using an Aurora spectral flow cytometer in the APC/iFluor® 750 specific R7-A channel.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Freadiuse-preactivated-apc-ifluor-750-tandem%2Ffigure-for-readiuse-preactivated-apc-ifluor-750-tandem_VNyHD.png&w=640&q=75)
![Flow cytometry analysis of whole blood cells stained with APC/iFluor® 750 anti-human CD4 antibody (Clone: SK3). The fluorescence signal was monitored using an Aurora spectral flow cytometer in the APC/iFluor® 750 specific R7-A channel.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Freadiuse-preactivated-apc-ifluor-750-tandem%2Ffigure-for-readiuse-preactivated-apc-ifluor-750-tandem_VNyHD.png&w=128&q=25)
![Flow cytometry analysis of PBMC stained with APC-iFluor® 750 anti-human CD3 *UCHT1* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the APC-iFluor® 750 specific R7-A channel.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Freadiuse-preactivated-apc-ifluor-750-tandem%2Ffigure-for-readiuse-preactivated-apc-ifluor-750-tandem_6nIVI.png&w=128&q=25)
![Spectrum for APC-iFluor 750](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Freadiuse-preactivated-apc-ifluor-750-tandem%2Ffigure-for-readiuse-preactivated-apc-ifluor-750-tandem_DJZsR.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-iFluor™ 750 Tandem
- Reconstitute pre-activated APC-iFluor™ 750 Tandem in 100 µL ddH2O to 10 mg/mL.
Note Reconstituted pre-activated APC-iFluor™ 750 Tandem could be stored at 4 °C for one month, kept from light. - Add APC-iFluor™ 750 Tandem directly to MTA-activated target Ab solution at the ratio of 130 µg APC-iFluor™ 750 Tandem/100 µg MTA-activated Ab.
- Rotate the mixture for 60 minutes at room temperature.
- The Ab/APC-iFluor™ 750 Tandem 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-iFluor™ 750 Tandem conjugates solution could be stored at 4 °C for up to two months, and kept from light. - (Optional) Ab/APC-iFluor™ 750 Tandem 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 APC-iFluor® 700 Tandem | 651 | 710 | 700000 |
ReadiUse™ Preactivated APC-iFluor® 800 Tandem | 651 | 819 | 700000 |
ReadiUse™ Preactivated PE-iFluor® 750 Tandem | 565 | 778 | 1960000 |
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