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.
![Our preactivated APC 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 (provided) to give the desired APC-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated APC 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-ifluor-800-tandem%2Ffigure-for-readiuse-preactivated-apc-ifluor-800-tandem_x2vVV.jpg&w=640&q=75)
![Our preactivated APC 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 (provided) to give the desired APC-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated APC 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-ifluor-800-tandem%2Ffigure-for-readiuse-preactivated-apc-ifluor-800-tandem_x2vVV.jpg&w=640&q=75)
![Our preactivated APC 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 (provided) to give the desired APC-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated APC 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-ifluor-800-tandem%2Ffigure-for-readiuse-preactivated-apc-ifluor-800-tandem_x2vVV.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™ 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