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ReadiLink™ xtra Rapid iFluor® 750 Antibody Labeling Kit *BSA-Compatible*

HL-60 cells were incubated with (red) or without (green) anti-human HLA-ABC (W6/32 mAb). Cells were then incubated with goat anti-mouse IgG labeled using the ReadiLink™ xtra Rapid iFluor® 750 Antibody Labeling Kit (Cat No. 1965). The fluorescence signal was monitored using an ACEA NovoCyte flow cytometer in the APC-Cy7 channel.
HL-60 cells were incubated with (red) or without (green) anti-human HLA-ABC (W6/32 mAb). Cells were then incubated with goat anti-mouse IgG labeled using the ReadiLink™ xtra Rapid iFluor® 750 Antibody Labeling Kit (Cat No. 1965). The fluorescence signal was monitored using an ACEA NovoCyte flow cytometer in the APC-Cy7 channel.
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Catalog Number1965
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Correction Factor (260 nm)0.044
Correction Factor (280 nm)0.039
Correction Factor (565 nm)0.0250
Correction Factor (650 nm)0.1413
Extinction coefficient (cm -1 M -1)2750001
Excitation (nm)757
Emission (nm)779
Quantum yield0.121
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


Correction Factor (260 nm)
0.044
Correction Factor (280 nm)
0.039
Correction Factor (565 nm)
0.0250
Correction Factor (650 nm)
0.1413
Extinction coefficient (cm -1 M -1)
2750001
Excitation (nm)
757
Emission (nm)
779
Quantum yield
0.121
ReadiLink™ xtra rapid antibody labeling kits require essentially only 2 simple mixing steps without a column purification needed. Preactivated iFluor® 750 used in this ReadiLink™ kit is quite stable and shows good reactivity and selectivity with antibodies. The kit has all the essential components for labeling ~2x50 ug antibody. Each of the two vials of preactivated iFluor® 750 dye provided in the kit is optimized for labeling ~50 µg antibody. ReadiLink™ xtra iFluor® 750 rapid antibody labeling kit provides a convenient and robust method to label monoclonal and polyclonal antibodies with the bright NIR fluorescent iFluor® 750 fluorophore. AAT Bioquest's iFluor® dyes are optimized for labeling proteins, in particular, antibodies. These dyes are bright, photostable and have minimal quenching on proteins. They can be well excited by the major laser lines of fluorescence instruments (e.g., 350, 405, 488, 555 and 633 nm).

 

readilinkworkflow

 

Figure 1. Overview of the ReadiLink™ xtra Rapid Antibody Labeling protocol. In just two simple steps, and with no purification necessary, covalently label microgram amounts of antibodies in under an hour.

Components


Component A: Preactivated iFluor® 7502 vials
Component B: Reaction Buffer1 vial (20 µL)
Component C: TQ™-Dyed Quench Buffer1 vial (20 µL)

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) with 0.1 to 0.5 % will 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


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.044
Correction Factor (280 nm)0.039
Correction Factor (565 nm)0.0250
Correction Factor (650 nm)0.1413
Extinction coefficient (cm -1 M -1)2750001
Excitation (nm)757
Emission (nm)779
Quantum yield0.121

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
ReadiLink™ xtra Rapid iFluor® 350 Antibody Labeling Kit *BSA-Compatible*3454502000010.9510.830.23
ReadiLink™ xtra Rapid iFluor® 488 Antibody Labeling Kit *BSA-Compatible*4915167500010.910.210.11
ReadiLink™ xtra Rapid iFluor® 555 Antibody Labeling Kit *BSA-Compatible*55757010000010.6410.230.14
ReadiLink™ xtra Rapid iFluor® 594 Antibody Labeling Kit *BSA-Compatible*58860418000010.5310.050.04
ReadiLink™ xtra Rapid iFluor® 647 Antibody Labeling Kit *BSA-Compatible*65667025000010.2510.030.03

References


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Journal: Oncotarget (2018): 34972-34989
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Journal: ACS applied materials & interfaces (2018): 30071-30080
Phosphorothioate-Modified AP613-1 Specifically Targets GPC3 when Used for Hepatocellular Carcinoma Cell Imaging.
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Journal: Molecular therapy. Nucleic acids (2018): 376-386
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Journal: Quantitative imaging in medicine and surgery (2018): 151-160
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Authors: Saccomano, Mara and Dullin, Christian and Alves, Frauke and Napp, Joanna
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