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ReadiUse™ Preactivated APC-Cy7 Maleimide
APC-Cy7 is a popular tandem 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 reduced antibodies and other biomolecules that contain a thiol group. Our preactivated APC-Cy7 maleimide is prepared from the commonly used crosslinker SMCC, and ready to conjugate. 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.
Example protocol
SAMPLE EXPERIMENTAL PROTOCOL
Reduction of Antibody
- Prepare a fresh solution of 1.0 M DTT (15.4 mg/100 µL) in distilled water. Antibody solutions should be at 2 mg/mL or higher for best results. The reduction can be carried out in different buffers for example: MES, phosphate, and TRIS buffers (pH range 6 to 8). The antibody should be concentrated if less than 2 mg/mL.
- Add 2 µL of 1.0 M DTT stock per 100 µL of antibody solution and mix well. Let the antibody solution stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
- Purify the reduced antibody over a desalting column pre-equilibrated with 50 mM MES Buffer (pH=6.0-6.5) with 2 mM EDTA. (Desalting column: https://www.aatbio.com/products/readiuse-bio-gel-p-6-spin-column?unit=60500)
- Measure the Antibody concentration with Nanodrop. (Con. (mg/ml)= A280nm/1.4) Note: The reduced antibody is not stable; the conjugation reaction needs to be carried out the conjugation as soon as possible after purification.
Conjugate with ReadiUse™ Preactivated APC-Cy7 Maleimide
- Reconstitute ReadiUse™ Preactivated APC-Cy7 Maleimide in 100 µL ddH2O to 10 mg/mL. Note: Reconstituted ReadiUse™ Preactivated APC-Cy7 Maleimide are stable at 4 °C for one week, please kept it from light.
- Add reduced antibody to pre-activated APC-Cy7 directly to at the ratio of 130 µg APC-Cy7 /100 µg reduced antibody.
- Rotate the mixture for 60-120 mins at room temperature.
- After 60 minutes, block the free sulfhydryls on the antibody.
- Prepare a fresh solution of 10 mg/mL NEM in DMSO; add 3.4 µL per mg of antibody and rotate for 20 minutes at room temperature.
Purification
- Antibody/APC-Cy7 conjugate could be further purified through size exclusion chromatography to get best performance. Note: The Antibody/APC-Cy7 conjugate solution is recommended to be stored at 2~8 °C and kept from light.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) |
| ReadiUse™ Preactivated APC-Cy7 Tandem | 651 | 779 | 700000 |
| ReadiUse™ Preactivated PE-Cy7 Maleimide | 565 | 778 | 1960000 |
| ReadiUse™ Preactivated PerCP-Cy7 Maleimide | 757 | 783 | 350000 |
References
View all 17 references: Citation Explorer
CD4+ T cells and natural killer cells: Biomarkers for hepatic fibrosis in human immunodeficiency virus/hepatitis C virus-coinfected patients.
Authors: Laufer, Natalia and Ojeda, Diego and Polo, María Laura and Martinez, Ana and Pérez, Héctor and Turk, Gabriela and Cahn, Pedro and Zwirner, Norberto Walter and Quarleri, Jorge
Journal: World journal of hepatology (2017): 1073-1080
Authors: Laufer, Natalia and Ojeda, Diego and Polo, María Laura and Martinez, Ana and Pérez, Héctor and Turk, Gabriela and Cahn, Pedro and Zwirner, Norberto Walter and Quarleri, Jorge
Journal: World journal of hepatology (2017): 1073-1080
Quantification of mitochondrial reactive oxygen species in living cells by using multi-laser polychromatic flow cytometry.
Authors: De Biasi, Sara and Gibellini, Lara and Bianchini, Elena and Nasi, Milena and Pinti, Marcello and Salvioli, Stefano and Cossarizza, Andrea
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2016): 1106-1110
Authors: De Biasi, Sara and Gibellini, Lara and Bianchini, Elena and Nasi, Milena and Pinti, Marcello and Salvioli, Stefano and Cossarizza, Andrea
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2016): 1106-1110
Presence of CD34(+)CD38(-)CD58(-) leukemia-propagating cells at diagnosis identifies patients at high risk of relapse with Ph chromosome-positive ALL after allo-hematopoietic SCT.
Authors: Kong, Y and Xu, L-P and Liu, Y-R and Qin, Y-Z and Sun, Y-Q and Wang, Y and Jiang, H and Jiang, Q and Chen, H and Chang, Y-J and Huang, X-J
Journal: Bone marrow transplantation (2015): 348-53
Authors: Kong, Y and Xu, L-P and Liu, Y-R and Qin, Y-Z and Sun, Y-Q and Wang, Y and Jiang, H and Jiang, Q and Chen, H and Chang, Y-J and Huang, X-J
Journal: Bone marrow transplantation (2015): 348-53
Analysis of Populations of Memory T-Helper Cells Expressing CXCR3 and CCR6 Chemokine Receptors in Peripheral Blood of Patients with Chronic Viral Hepatitis C.
Authors: Elezov, D S and Kudryavtsev, I V and Arsent'ev, N A and Basin, V V and Esaulenko, E V and Semenov, A V and Totolyan, A A
Journal: Bulletin of experimental biology and medicine (2015): 238-42
Authors: Elezov, D S and Kudryavtsev, I V and Arsent'ev, N A and Basin, V V and Esaulenko, E V and Semenov, A V and Totolyan, A A
Journal: Bulletin of experimental biology and medicine (2015): 238-42
A flow cytometric method for the analysis of macrophages in the vascular wall.
Authors: Moore, Jeffrey P and Sakkal, Samy and Bullen, Michelle L and Kemp-Harper, Barbara K and Ricardo, Sharon D and Sobey, Christopher G and Drummond, Grant R
Journal: Journal of immunological methods (2013): 33-43
Authors: Moore, Jeffrey P and Sakkal, Samy and Bullen, Michelle L and Kemp-Harper, Barbara K and Ricardo, Sharon D and Sobey, Christopher G and Drummond, Grant R
Journal: Journal of immunological methods (2013): 33-43
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