ReadiUse™ Preactivated PE-iFluor® 700 Tandem

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/iFlour® 700 anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the PE/iFluor® 700 specific B10-A channel.

Flow cytometry analysis of whole blood stained with PE-iFluor® 700 anti-human CD19 *HIB19* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the PE-iFluor® 700 specific B10-A channel.

Our preactivated PE-iFluor 700 tandem 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-iFluor® 700 Tandem (provided) to give the desired PE-iFluor® 700 Tandem-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated PE-iFluor® 700 Tandem reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.

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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

Molecular weight	N/A
Solvent	Water

Spectral properties

Absorbance (nm)	566
Extinction coefficient (cm ^-1 M ^-1)	1960000
Excitation (nm)	565
Emission (nm)	708

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

Molecular weight

N/A

Absorbance (nm)

566

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

1960000

Excitation (nm)

565

Emission (nm)

708

PE-Alexa Fluor® 700 is a popular color used in flow cytometry. Its primary absorption peak is at 565 nm with emission peak at 720 nm. AAT Bioquest offers this preactivated PE-iFluor 700™ as a superior replacement to the popular PE- Alexa Fluor® 700 tandem. Compared to PE-Alexa Fluor® 700 tandem, PE-iFluor 700 is brighter with better FRET efficiency. It is used to facilitate the PE-Alexa Fluor® 700 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated PE-iFluor 700 tandem is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based PE-Alexa Fluor® 700 tandem conjugation chemistry with better flow cytometry performance. In addition, our preactivated PE-iFluor 700 tandem 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-iFluor™ 700 Tandem 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-iFluor™ 700 Tandem (provided) to give the desired PE-iFluor™ 700 Tandem-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-iFluor™ 700 Tandem

Reconstitute pre-activated PE-iFluor™ 700 Tandem in 100 µL ddH₂O to 10 mg/mL.
Note Reconstituted pre-activated PE-iFluor™ 700 Tandem is not stable and can not be stored for more than one month.
Add pre-activated PE-iFluor™ 700 Tandem directly to MTA-activated target Ab solution at the ratio of 300 µg PE-iFluor™ 700 Tandem/100 µg MTA-activated Ab.
Rotate the mixture for 1 - 2 hours at room temperature.
The Ab/PE-iFluor™ 700 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.
Note The Ab/PE-iFluor™ 700 Tandem can be stored at 4 °C for two months.
Optional: Ab/PE-iFluor™ 700 Tandem can be further purified through size exclusion chromatography to get better performance.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Absorbance (nm)	566
Extinction coefficient (cm ^-1 M ^-1)	1960000
Excitation (nm)	565
Emission (nm)	708

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)
ReadiUse™ Preactivated PE-iFluor® 647 Tandem	565	666	1960000
ReadiUse™ Preactivated PE-iFluor® 750 Tandem	565	778	1960000
ReadiUse™ Preactivated PE-iFluor® 594 Tandem	565	606	1960000
ReadiUse™ Preactivated PE-iFluor® 660 Tandem	565	695	1960000
ReadiUse™ Preactivated APC-iFluor® 700 Tandem	651	710	700000

Images

Figure 1. 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/iFlour® 700 anti-human CD4 *SK3* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the PE/iFluor® 700 specific B10-A channel.

Figure 2. Flow cytometry analysis of whole blood stained with PE-iFluor® 700 anti-human CD19 *HIB19* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the PE-iFluor® 700 specific B10-A channel.

Figure 3. Our preactivated PE-iFluor 700 tandem 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-iFluor® 700 Tandem (provided) to give the desired PE-iFluor® 700 Tandem-antibody conjugate in much higher yield than the SMCC chemistry. In addition our preactivated PE-iFluor® 700 Tandem reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.

References

View all 7 references: Citation Explorer

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Journal: Biomedical optics express (2018): 2229-2239

An enzymatically-sensitized sequential and concentric energy transfer relay self-assembled around semiconductor quantum dots.
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Journal: Nanoscale (2015): 7603-14

Noninvasive and quantitative assessment of in vivo fetomaternal interface angiogenesis using RGD-based fluorescence.
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Journal: BioMed research international (2014): 309082

Multicolor detection of rare tumor cells in blood using a novel flow cytometry-based system.
Authors: Watanabe, Masaru and Uehara, Yuri and Yamashita, Namiko and Fujimura, Yuu and Nishio, Kaori and Sawada, Takeshi and Takeda, Kazuo and Koizumi, Fumiaki and Koh, Yasuhiro
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2014): 206-13

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Authors: Keramidas, M and Josserand, V and Righini, C A and Wenk, C and Faure, C and Coll, J L
Journal: The British journal of surgery (2010): 737-43

Homogeneous TR-FRET high-throughput screening assay for calcium-dependent multimerization of sorcin.
Authors: Appelblom, Heidi and Nurmi, Jussi and Soukka, Tero and Pasternack, Michael and Penttilä, Kai E and Lövgren, Timo and Niemelä, Pauliina
Journal: Journal of biomolecular screening (2007): 842-8

Analysis of hollow-core photonic bandgap fibers for evanescent wave biosensing.
Authors: Sun, Jian and Chan, Chi-Chiu and Zhang, Yi-Fan and Shum, Ping
Journal: Journal of biomedical optics : 054048