ReadiUse™ Preactivated PE

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 flow cytometer in the PE specific B6-A channel.

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Physical properties

Solvent

Water

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	1960000
Excitation (nm)	565
Emission (nm)	574
Quantum yield	0.82

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Refrigerated (2-8 °C); Minimize light exposure
UNSPSC	12171501

Overview SDS Protocol

Extinction coefficient (cm ^-1 M ^-1)

1960000

Excitation (nm)

565

Emission (nm)

574

Quantum yield

0.82

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.

Components

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

Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	1960000
Excitation (nm)	565
Emission (nm)	574
Quantum yield	0.82

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)
ReadiUse™ Preactivated APC	651	660	700000
ReadiUse™ Preactivated PerCP	482	678	350000

Images

Figure 1. 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.

Figure 2. 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 flow cytometer in the PE specific B6-A channel.

References

View all 96 references: Citation Explorer

Detection of LC3-Associated Phagocytosis (LAP).
Authors: Martinez, Jennifer
Journal: Current protocols in cell biology (2020): e104

CoViD-19 Immunopathology and Immunotherapy.
Authors: Chiappelli, Francesco and Khakshooy, Allen and Greenberg, Gillian
Journal: Bioinformation (2020): 219-222

High-Resolution Imaging Flow Cytometry Reveals Impact of Incubation Temperature on Labeling of Extracellular Vesicles with Antibodies.
Authors: Tertel, Tobias and Bremer, Michel and Maire, Cecile and Lamszus, Katrin and Peine, Sven and Jawad, Rim and Andaloussi, Samir E L and Giebel, Bernd and Ricklefs, Franz L and Görgens, André
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)

Automated Motion Tracking and Data Extraction for Red Blood Cell Biomechanics.
Authors: Kumar, Arun and Schmidt, Brendan R and Sanchez, Zyrina Alura C and Yazar, Feyza and Davis, Ronald W and Ramasubramanian, Anand K and Saha, Amit K
Journal: Current protocols in cytometry (2020): e75

Seeing the Confetti Colors in a New Light Utilizing Flow Cytometry and Imaging Flow Cytometry.
Authors: Hagert, Cecilia Fahlquist and Bohn, Anja Bille and Wittenborn, Thomas R and Degn, Søren E
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)

High-Dimensional Data Analysis Algorithms Yield Comparable Results for Mass Cytometry and Spectral Flow Cytometry Data.
Authors: Ferrer-Font, Laura and Mayer, Johannes U and Old, Samuel and Hermans, Ian F and Irish, Jonathan and Price, Kylie M
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)

Development of Automated Microscopy-Assisted High-Content Multiparametric Assays for Cell Cycle Staging and Foci Quantitation.
Authors: Frölich, Sonja and Robker, Rebecca and Russell, Darryl
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020): 378-393

The Challenge of Distinguishing Cell-Cell Complexes from Singlet Cells in Non-Imaging Flow Cytometry and Single-Cell Sorting.
Authors: Burel, Julie G and Pomaznoy, Mikhail and Lindestam Arlehamn, Cecilia S and Seumois, Gregory and Vijayanand, Pandurangan and Sette, Alessandro and Peters, Bjoern
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)

Flow Cytometric Monitoring of Dynamic Cytosolic Calcium, Sodium, and Potassium Fluxes Following Platelet Activation.
Authors: Aliotta, Alessandro and Bertaggia Calderara, Debora and Alberio, Lorenzo
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2020)

Thirty years of translational research in Mobility Medicine: Collection of abstracts of the 2020 Padua Muscle Days.
Authors: Carraro, Ugo
Journal: European journal of translational myology (2020): 8826