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AAT Bioquest

ReadiLink™ Rapid XFD750 Antibody Labeling Kit *XFD750 Same Structure to Alexa Fluor™ 750*

XFD750 is manufactured by AAT Bioquest, and it has the same chemical structure as Alexa Fluor® 750 (Alexa Fluor® is the trademark of ThermoFisher). Readilink™ protein labeling technology is the most robust and convenient tool for preparing fluorescent antibody conjugates for fluorescence imaging and flow cytometry applications. ReadiLink™ Rapid XFD750 Antibody Labeling Kit provides the most convenient tool for making XFD750-labeled antibody conjugates. XFD750 dye used in this ReadiLink™ kit is reasonably stable and shows good reactivity and selectivity with protein amino groups. The kit has all the essential components for labeling ~2x50 ug antibody. Each of the two vials of XFD750 dye provided in the kit is optimized for labeling ~50 µg antibody. ReadiLink™ Rapid XFD750 Antibody Labeling Kit requires minimal hands-on time. The prepared XFD750 conjugates (with the kit) are ready to use for fluorescence imaging and flow cytometry applications without further purifications needed. It provides the most convenient method to prepare XFD750-labeled antibodies.

Example protocol

AT A GLANCE

Important

Warm all the components and centrifuge the vials briefly before opening them. Immediately prepare the necessary solutions before starting your conjugation. The following protocol is a recommendation.

PREPARATION OF WORKING SOLUTION

Protein working solution (Solution A)

For labeling 50 µg of protein (assuming the target protein concentration is 1 mg/mL), mix 5 µL (10% of the total reaction volume) of Reaction Buffer (Component B) with 50 µL of the target protein solution.

Note: If you have a different protein concentration, adjust the protein volume accordingly to make ~50 µg of protein available for your labeling reaction.

Note: For labeling 100 µg of protein (assuming the target protein concentration is 1 mg/mL), mix 10 µL (10% of the total reaction volume) of Reaction Buffer (Component B) with 100 µL of the target protein solution.

Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2 - 7.4; if the protein is dissolved in glycine buffer, it must be dialyzed against 1X PBS, pH 7.2 - 7.4, or use Amicon Ultra-0.5, Ultracel-10 Membrane, 10 kDa (Cat. UFC501008 from Millipore) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.

Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.

Note: A final protein concentration range of 1-2 mg/mL is recommended for optimal labeling efficiency, with a significantly reduced conjugation efficiency at less than 1 mg/mL.

SAMPLE EXPERIMENTAL PROTOCOL

Run conjugation reaction
  1. Add the protein working solution (Solution A) to ONE vial of labeling dye (Component A), and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

    Note: If labeling 100 µg of protein, use both vials (Component A) of labeling dye by dividing the 100 µg of protein into 2 x 50 µg of protein and reacting each 50 µg of protein with one vial of labeling dye. Then combine both vials for the next step. 

  2. Keep the conjugation reaction mixture at room temperature for 30-60 minutes.

    Note: The conjugation reaction mixture can be rotated or shaken for a longer time if desired. 

Stop Conjugation reaction
  1. Add 5 µL (for 50 µg protein) or 10 µL (for 100 µg protein) which is 10% of the total reaction volume of TQ™-Dyed Quench Buffer (Component C) into the conjugation reaction mixture; mix well.

  2. Incubate at room temperature for 10 minutes. The labeled protein (antibody) is now ready to use.

Storage of Protein Conjugate

The protein conjugate should be stored at > 0.5 mg/mL in the presence of a carrier protein (e.g., 0.1% bovine serum albumin). For longer storage, the protein conjugates could be lyophilized or divided into single-used aliquots and stored at ≤ –20°C. 

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (280 nm)Correction Factor (260 nm)
ReadiLink™ Rapid FITC Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*491516730000.920.35-
ReadiLink™ Rapid Cy3 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*55556915000010.1510.0730.07
ReadiLink™ Rapid Cy5 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*65167025000010.271, 0.420.030.02
ReadiLink™ Rapid Cy7 Antibody Labeling Kit *Microscale Optimized for Labeling 50 μg Antibody Per Reaction*7567792500000.30.0360.05
ReadiLink™ Rapid XFD488 Antibody Labeling Kit *XFD488 Same Structure to Alexa Fluor™ 488*499520710000.9210.110.30
ReadiLink™ Rapid XFD555 Antibody Labeling Kit *XFD555 Same Structure to Alexa Fluor™ 555*5535681500000.110.080.08
ReadiLink™ Rapid XFD594 Antibody Labeling Kit *XFD594 Same Structure to Alexa Fluor™ 594*590618900000.6610.560.43
ReadiLink™ Rapid XFD647 Antibody Labeling Kit *XFD647 Same Structure to Alexa Fluor™ 647*6506712390000.3310.030.00
ReadiLink™ Rapid FITC Antibody Labeling Kit *Production Scale*491516730000.920.35-
ReadiLink™ Rapid Cy3 Antibody Labeling Kit *Production Scale*55556915000010.1510.0730.07
ReadiLink™ Rapid Cy5 Antibody Labeling Kit *Production Scale*65167025000010.271, 0.420.030.02
ReadiLink™ Rapid XFD488 Antibody Labeling Kit *Production Scale*499520710000.9210.110.30
ReadiLink™ Rapid XFD555 Antibody Labeling Kit *Production Scale*5535681500000.110.080.08
ReadiLink™ Rapid XFD594 Antibody Labeling Kit *Production Scale*590618900000.6610.560.43
ReadiLink™ Rapid Cy7 Antibody Labeling Kit *Production Scale*7567792500000.30.0360.05
ReadiLink™ Rapid XFD647 Antibody Labeling Kit *Production Scale*6506712390000.3310.030.00
Show More (7)

References

View all 28 references: Citation Explorer
Challenging a Preconception: Optoacoustic Spectrum Differs from the Optical Absorption Spectrum of Proteins and Dyes for Molecular Imaging.
Authors: Fuenzalida Werner, Juan Pablo and Huang, Yuanhui and Mishra, Kanuj and Janowski, Robert and Vetschera, Paul and Heichler, Christina and Chmyrov, Andriy and Neufert, Clemens and Niessing, Dierk and Ntziachristos, Vasilis and Stiel, Andre C
Journal: Analytical chemistry (2020)
CD24-targeted intraoperative fluorescence image-guided surgery leads to improved cytoreduction of ovarian cancer in a preclinical orthotopic surgical model.
Authors: Kleinmanns, Katrin and Fosse, Vibeke and Davidson, Ben and de Jalón, Elvira García and Tenstad, Olav and Bjørge, Line and McCormack, Emmet
Journal: EBioMedicine (2020): 102783
Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging.
Authors: Duranti, Claudia and Carraresi, Laura and Sette, Angelica and Stefanini, Matteo and Lottini, Tiziano and Crescioli, Silvia and Crociani, Olivia and Iamele, Luisa and De Jonge, Hugo and Gherardi, Ermanno and Arcangeli, Annarosa
Journal: Oncotarget (2018): 34972-34989
Enhanced Release of Molecules upon Ultraviolet (UV) Light Irradiation from Photoresponsive Hydrogels Prepared from Bifunctional Azobenzene and Four-Arm Poly(ethylene glycol).
Authors: Rastogi, Shiva K and Anderson, Hailee E and Lamas, Joseph and Barret, Scott and Cantu, Travis and Zauscher, Stefan and Brittain, William J and Betancourt, Tania
Journal: ACS applied materials & interfaces (2018): 30071-30080
Phosphorothioate-Modified AP613-1 Specifically Targets GPC3 when Used for Hepatocellular Carcinoma Cell Imaging.
Authors: Dong, Lili and Zhou, Hongxin and Zhao, Menglong and Gao, Xinghui and Liu, Yang and Liu, Dongli and Guo, Wei and Hu, Hongwei and Xie, Qian and Fan, Jia and Lin, Jiang and Wu, Weizhong
Journal: Molecular therapy. Nucleic acids (2018): 376-386
Page updated on November 9, 2024

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Catalog Number1279
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Spectral properties

Correction Factor (260 nm)

0.00

Correction Factor (280 nm)

0.04

Extinction coefficient (cm -1 M -1)

240000

Excitation (nm)

752

Emission (nm)

776

Quantum yield

0.121

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

Components