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ReadiUse™ Preactivated APC-iFluor® 800 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® 800 Tandem is a unique fluorophore with an IR emission developed for spectral flow cytometry. Its emission spectrum does not overlap with any of the common fluorophores, making a unique color for multicolor applications in flow cytometry. ReadiUse™ Preactivated APC-iFluor® 800 Tandem is an activated APC protein, and can be easily conjugated to antibodies with much higher conjugation yield than the conventional APC tandems.
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™ 800 Tandem
- Reconstitute pre-activated APC-iFluor™ 800 Tandem in 100 µL ddH2O to 10 mg/mL.
Note Reconstituted pre-activated APC-iFluor™ 800 Tandem could be stored at 4 °C for one month, kept from light. - Add APC-iFluor™ 800 Tandem directly to MTA-activated target Ab solution at the ratio of 130 µg APC-iFluor™ 800 Tandem/100 µg MTA-activated Ab.
- Rotate the mixture for 60 minutes at room temperature.
- The Ab/APC-iFluor™ 800 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™ 800 Tandem conjugates solution could be stored at 4 °C for up to two months, and kept from light. - (Optional) Ab/APC-iFluor™ 800 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® 750 Tandem | 651 | 793 | 700000 |
References
View all 2 references: Citation Explorer
Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.
Authors: Le Roy, Christine and Varin-Blank, Nadine and Ajchenbaum-Cymbalista, Florence and Letestu, Rémi
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2009): 882-90
Authors: Le Roy, Christine and Varin-Blank, Nadine and Ajchenbaum-Cymbalista, Florence and Letestu, Rémi
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2009): 882-90
Cy7PE and Cy7APC: bright new probes for immunofluorescence.
Authors: Roederer, M and Kantor, A B and Parks, D R and Herzenberg, L A
Journal: Cytometry (1996): 191-7
Authors: Roederer, M and Kantor, A B and Parks, D R and Herzenberg, L A
Journal: Cytometry (1996): 191-7
Page updated on December 17, 2024
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