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ReadiLink™ xtra Rapid iFluor® 594 Antibody Labeling Kit *BSA-Compatible*

ReadiLink™ xtra rapid antibody labeling kits require essentially only 2 simple mixing steps without a column purification needed. Preactivated iFluor® 594 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® 594 dye provided in the kit is optimized for labeling ~50 µg antibody. ReadiLink™ xtra iFluor® 594 rapid antibody labeling kit provides a convenient and robust method to label monoclonal and polyclonal antibodies with the bright red fluorescent iFluor® 594 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.

Example protocol

AT A GLANCE

Important

Warm all the components and centrifuge the vials briefly before opening, and immediately prepare the required solutions before starting your conjugation. The following protocol is for 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        For optimal labeling efficiency, a final protein concentration range of 1 - 2 mg/mL is recommended, 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 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 (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® 647 Antibody Labeling Kit *BSA-Compatible*65667025000010.2510.030.03
ReadiLink™ xtra Rapid iFluor® 750 Antibody Labeling Kit *BSA-Compatible*75777927500010.1210.0440.039

References

View all 8 references: Citation Explorer
Bioorthogonal click chemistry for fluorescence imaging of choline phospholipids in plants.
Authors: Paper, Janet M and Mukherjee, Thiya and Schrick, Kathrin
Journal: Plant methods (2018): 31
WGA-Alexa transsynaptic labeling in the phrenic motor system of adult rats: Intrapleural injection versus intradiaphragmatic injection.
Authors: Buttry, Janelle L and Goshgarian, Harry G
Journal: Journal of neuroscience methods (2015): 137-45
Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.
Authors: Liao-Chan, Sindy and Daine-Matsuoka, Barbara and Heald, Nathan and Wong, Tiffany and Lin, Tracey and Cai, Allen G and Lai, Michelle and D'Alessio, Joseph A and Theunissen, Jan-Willem
Journal: PloS one (2015): e0124708
Site-specific labeling of proteins for single-molecule FRET measurements using genetically encoded ketone functionalities.
Authors: Lemke, Edward A
Journal: Methods in molecular biology (Clifton, N.J.) (2011): 3-15
Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM(197) conjugate vaccine co-localizes with MHC II on the antigen processing cell surface.
Authors: Lai, Zengzu and Schreiber, John R
Journal: Vaccine (2009): 3137-44
Page updated on October 4, 2024

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Catalog Number1960
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Spectral properties

Absorbance (nm)

587

Correction Factor (260 nm)

0.05

Correction Factor (280 nm)

0.04

Extinction coefficient (cm -1 M -1)

2000001

Excitation (nm)

587

Emission (nm)

603

Quantum yield

0.531

Storage, safety and handling

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

Components

Immunofluorescence staining of tubulin in HeLa cells. HeLa cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and blocked. Cells were then incubated with mouse anti-tubulin monoclonal antibody and stained with a goat anti-mouse IgG labeled using the ReadiLink™ xtra Rapid iFluor® 594 Antibody Labeling Kit (Cat No. 1960).
Immunofluorescence staining of tubulin in HeLa cells. HeLa cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and blocked. Cells were then incubated with mouse anti-tubulin monoclonal antibody and stained with a goat anti-mouse IgG labeled using the ReadiLink™ xtra Rapid iFluor® 594 Antibody Labeling Kit (Cat No. 1960).
Immunofluorescence staining of tubulin in HeLa cells. HeLa cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and blocked. Cells were then incubated with mouse anti-tubulin monoclonal antibody and stained with a goat anti-mouse IgG labeled using the ReadiLink™ xtra Rapid iFluor® 594 Antibody Labeling Kit (Cat No. 1960).