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United States of America
Telephone:1-408-733-1055
Fax:1-408-733-1304
Email:sales@aatbio.com
Purchase Order:sales@aatbio.com

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ReadiUse™ Preactivated PE-iFluor® 700 Tandem

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.

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
  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-iFluor™ 700 Tandem
  1. 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.
  2. 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.
  3. Rotate the mixture for 1 - 2 hours at room temperature.
  4. 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.
  5. Optional: Ab/PE-iFluor™ 700 Tandem 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)
ReadiUse™ Preactivated PE-iFluor® 647 Tandem5656661960000
ReadiUse™ Preactivated PE-iFluor® 750 Tandem5657781960000
ReadiUse™ Preactivated PE-iFluor® 594 Tandem5656061960000
ReadiUse™ Preactivated PE-iFluor® 660 Tandem5656951960000
ReadiUse™ Preactivated APC-iFluor® 700 Tandem651710700000

References

View all 7 references: Citation Explorer
Performance of optoacoustic and fluorescence imaging in detecting deep-seated fluorescent agents.
Authors: Chen, Zhenyue and Deán-Ben, Xosé Luís and Gottschalk, Sven and Razansky, Daniel
Journal: Biomedical optics express (2018): 2229-2239
An enzymatically-sensitized sequential and concentric energy transfer relay self-assembled around semiconductor quantum dots.
Authors: Samanta, Anirban and Walper, Scott A and Susumu, Kimihiro and Dwyer, Chris L and Medintz, Igor L
Journal: Nanoscale (2015): 7603-14
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
Noninvasive and quantitative assessment of in vivo fetomaternal interface angiogenesis using RGD-based fluorescence.
Authors: Keramidas, M and Lavaud, J and Sergent, F and Hoffmann, P and Brouillet, S and Feige, J-J and Coll, J-L and Alfaidy, N
Journal: BioMed research international (2014): 309082
Intraoperative near-infrared image-guided surgery for peritoneal carcinomatosis in a preclinical experimental model.
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
Page updated on December 3, 2024

Ordering information

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Unit size
Catalog Number2585
Quantity
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Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

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

Storage

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

Components

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 spectral flow cytometer in the PE/iFluor® 700 specific B10-A channel.
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 spectral flow cytometer in the PE/iFluor® 700 specific B10-A channel.
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 spectral 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 spectral 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.

Documents

pdfSafety data sheet
pdfProduct protocol
pdfCertificate of analysis
Fluorescence Activated Cell Sorting (FACS)
PE and APC
Spectral Flow Cytometry
ReadiUse™ Preactivated APC-iFluor® 750 Tandem
ReadiUse™ ABTS Substrate Solution *Optimized for ELISA Assays with HRP Conjugates*
ReadiUse™ TMB Substrate Solution *Optimized for ELISA Assays with HRP Conjugates*