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ReadiUse™ Preactivated PE
R-Phycoerythrin (PE) is isolated from red algae. Its primary absorption peak is at 565 nm with secondary peaks at 496 and 545 nm. AAT Bioquest offers this preactivated PE to facilitate the PE conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated PE is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated PE 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.
Example protocol
AT A GLANCE
Important PE was premodified with our Buccutite™ FOL. Your antibody (or other proteins) is modified with our Buccutite™ MTA to give MTA-modified protein. The MTA-modified protein readily reacts with FOL-modified PE (provided) to give the desired PE-antibody conjugate.
SAMPLE EXPERIMENTAL PROTOCOL
Preparation of pre-activated Antibody with Buccutite™ MTA
- Reconstitute Buccutite™ MTA in DMSO at ~10 mg/mL.
Note Store unused MTA at -20 °C; it can be used for up to two freeze and thaw cycles. - Prepare target antibody (Ab) in pH = 8.5 - 9.0 buffer at a concentration above 1 mg/ml.
- Add the MTA to Ab solution at the ratio of 8 - 10 µg MTA/100 µg Ab.
- Mix well and react at room temperature for 60 minutes, rotating during the reaction.
- Purify the reaction mixture with a desalting column to remove any unreacted MTA. Exchange the buffer to PBS or another buffer of your choice.
- Collect the MTA-activated Ab. Estimate the concentration by 70% yield of the original starting amount.
Conjugate with Pre-activated PE
- Reconstitute pre-activated PE in 100 µL ddH2O to 10 mg/mL.
Note Reconstituted pre-activated PE is not stable and can not be stored for more than one month. - Add pre-activated PE directly to MTA-activated target Ab solution at the ratio of 300 µg PE/100 µg MTA-activated Ab.
- Rotate the mixture for 1 - 2 hours at room temperature.
- The Ab/PE 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.
Note The Ab/PE can be stored at 4 °C for two months. - Optional: Ab/PE can be further purified through size exclusion chromatography to get better performance.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Correction Factor (280 nm) |
| ReadiUse™ Preactivated APC | 651 | 660 | 730000 | 0.195 |
| ReadiUse™ Preactivated PerCP | 477 | 678 | 406000 | 0.22 |
References
View all 96 references: Citation Explorer
Imaging Flow Cytometry for Phylogenetic and MorphologicallyBased Functional Group Clustering of a Natural Phytoplankton Community over 1 Year in an Urban Pond.
Authors: Dunker, Susanne
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)
Authors: Dunker, Susanne
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)
In Vivo Photodynamic Therapy With a Lipophilic Zinc(II) Phthalocyanine Inhibits Colorectal Cancer and Induces a Th1/CD8 Antitumor Immune Response.
Authors: Chiarante, Nicolás and Duhalde Vega, Maite and Valli, Federico and Zotta, Elsa and Daghero, Hellen and Basika, Tatiana and Bollati-Fogolin, Mariela and García Vior, María C and Marino, Julieta and Roguin, Leonor P
Journal: Lasers in surgery and medicine (2020)
Authors: Chiarante, Nicolás and Duhalde Vega, Maite and Valli, Federico and Zotta, Elsa and Daghero, Hellen and Basika, Tatiana and Bollati-Fogolin, Mariela and García Vior, María C and Marino, Julieta and Roguin, Leonor P
Journal: Lasers in surgery and medicine (2020)
Purification of Live Stem-Cell-Derived Islet Lineage Intermediates.
Authors: Rasouli, Niloofar and Melton, Douglas A and Alvarez-Dominguez, Juan R
Journal: Current protocols in stem cell biology (2020): e111
Authors: Rasouli, Niloofar and Melton, Douglas A and Alvarez-Dominguez, Juan R
Journal: Current protocols in stem cell biology (2020): e111
Application Settings versus Fixed Photomultiplier Tube Voltages for Optimal Cytometer Stability over Time, and Effect on Reusing a Compensation Matrix.
Authors: Andersen, Morten Nørgaard and Skovbo, Anni and Petersen, Charlotte Christie and Hokland, Marianne
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)
Authors: Andersen, Morten Nørgaard and Skovbo, Anni and Petersen, Charlotte Christie and Hokland, Marianne
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)
Improved Flow Cytometric Light Scatter Detection of Submicron-Sized Particles by Reduction of Optical Background Signals.
Authors: Arkesteijn, Ger J A and Lozano-Andrés, Estefanía and Libregts, Sten F W M and Wauben, Marca H M
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)
Authors: Arkesteijn, Ger J A and Lozano-Andrés, Estefanía and Libregts, Sten F W M and Wauben, Marca H M
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)
Page updated on December 17, 2024
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