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AAT Bioquest

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
  1. 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.
  2. Prepare target antibody (Ab) in pH = 8.5 - 9.0 buffer at a concentration above 1 mg/ml.
  3. Add the MTA to Ab solution at the ratio of 8 - 10 µg MTA/100 µg Ab.
  4. Mix well and react at room temperature for 60 minutes, rotating during the reaction.
  5. Purify the reaction mixture with a desalting column to remove any unreacted MTA. Exchange the buffer to PBS or another buffer of your choice.
  6. Collect the MTA-activated Ab. Estimate the concentration by 70% yield of the original starting amount. 

Conjugate with Pre-activated PE
  1. 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.
  2. Add pre-activated PE directly to MTA-activated target Ab solution at the ratio of 300 µg PE/100 µg MTA-activated Ab.
  3. Rotate the mixture for 1 - 2 hours at room temperature.
  4. 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.
  5. Optional: Ab/PE can be further purified through size exclusion chromatography to get better performance. 

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Correction Factor (280 nm)
ReadiUse™ Preactivated APC6516607300000.195
ReadiUse™ Preactivated PerCP4776784060000.22

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)
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)
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
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)
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)
Page updated on December 13, 2024

Ordering information

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Catalog Number2560
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Additional ordering information

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Physical properties

Solvent

Water

Spectral properties

Correction Factor (280 nm)

0.175

Extinction coefficient (cm -1 M -1)

1960000

Excitation (nm)

565

Emission (nm)

574

Quantum yield

0.82

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Refrigerated (2-8 °C); Minimize light exposure
UNSPSC12171501

Components

Our preactivated PE 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 PE (provided) to give the desired PE-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated PE reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
Our preactivated PE 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 PE (provided) to give the desired PE-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated PE reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
Our preactivated PE 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 PE (provided) to give the desired PE-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated PE reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.
Top) Spectral pattern was generated using a 4-laser spectral cytometer. Spatially offset lasers (355 nm, 405 nm, 488 nm, and 640 nm) were used to create four distinct emission profiles, then, when combined, yielded the overall spectral signature. Bottom) Flow cytometry analysis of PBMC stained with PE anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora spectral flow cytometer in the PE specific B6-A channel.