ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction

Although FITC is still the most popular fluorescent labeling dye for preparing green fluorescent bioconjugates, there are certain limitations with FITC, such as severe photobleaching for microscope imaging and pH-sensitive fluorescence. Protein conjugates prepared with iFluor® 488 dyes are far superior compared to conjugates of fluorescein derivatives such as FITC. iFluor® 488 conjugates are significantly brighter than fluorescein conjugates and are much more photostable. Additionally, the fluorescence of iFluor® 488 is not affected by pH (4-10). This pH insensitivity is a major improvement over fluorescein, which emits its maximum fluorescence only at pH above 9. ReadiLink™ labeling kits essentially only require 2 simple mixing steps without a column purification needed. iFluor® 488 SE 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 iFluor® 488 dye provided in the kit is optimized for labeling ~50 µg antibody. iFluor® 488 SE protein labeling kit provides a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the iFluor® 488 SE.

Figure 1. Overview of the ReadiLink™ 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) or gelatin will not 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

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.
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

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.
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

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Product family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
ReadiLink™ Rapid iFluor® 350 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	345	450	20000¹	0.95¹	0.83	0.23
ReadiLink™ Rapid iFluor® 555 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	557	570	100000¹	0.64¹	0.23	0.14
ReadiLink™ Rapid iFluor® 594 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	587	603	200000¹	0.53¹	0.05	0.04
ReadiLink™ Rapid iFluor® 647 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	656	670	250000¹	0.25¹	0.03	0.03
ReadiLink™ Rapid iFluor® 680 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	684	701	220000¹	0.23¹	0.097	0.094
ReadiLink™ Rapid iFluor® 700 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	690	713	220000¹	0.23¹	0.09	0.04
ReadiLink™ Rapid iFluor® 750 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	757	779	275000¹	0.12¹	0.044	0.039
ReadiLink™ Rapid iFluor® 633 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	640	654	250000¹	0.29¹	0.062	0.044
ReadiLink™ Rapid iFluor® 790 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	787	812	250000¹	0.13¹	0.1	0.09
ReadiLink™ Rapid iFluor® 350 Antibody Labeling Kit Production Scale	345	450	20000¹	0.95¹	0.83	0.23
ReadiLink™ Rapid iFluor® 555 Antibody Labeling Kit Production Scale	557	570	100000¹	0.64¹	0.23	0.14
ReadiLink™ Rapid iFluor® 594 Antibody Labeling Kit Production Scale	587	603	200000¹	0.53¹	0.05	0.04
ReadiLink™ Rapid iFluor® 647 Antibody Labeling Kit Production Scale	656	670	250000¹	0.25¹	0.03	0.03
ReadiLink™ Rapid iFluor® 750 Antibody Labeling Kit Production Scale	757	779	275000¹	0.12¹	0.044	0.039
Show More (5)

Citations

View all 3 citations: Citation Explorer

Deep Sequencing Analysis of the Eha-Regulated Transcriptome of Edwardsiella tarda Following Acidification
Authors: Gao, D and Liu, N and Li, Y and Zhang, Y and Liu, G and others, undefined
Journal: Metabolomics (Los Angel) (2017): 2153--0769

Suramin inhibits cullin-RING E3 ubiquitin ligases
Authors: Wu, Kenneth and Chong, Robert A and Yu, Qing and Bai, Jin and Spratt, Donald E and Ching, Kevin and Lee, Chan and Miao, Haibin and Tappin, Inger and Hurwitz, Jerard and others, undefined
Journal: Proceedings of the National Academy of Sciences (2016): E2011--E2018

Glycosaminoglycan mimicry by COAM reduces melanoma growth through chemokine induction and function
Authors: Piccard, Helene and Berghmans, Nele and Korpos, Eva and Dillen, Chris and Aelst, Ilse Van and Li, S and ra , undefined and Martens, Erik and Liekens, S and ra , undefined and Noppen, Sam and Damme, Jo Van and others, undefined
Journal: International Journal of Cancer (2012): E425--E436

References

View all 49 references: Citation Explorer

Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based beta-lactam probes
Authors: Sadhu KK, Mizukami S, Watanabe S, Kikuchi K.
Journal: Mol Biosyst (2011): 1766

Visualizing dengue virus through Alexa Fluor labeling
Authors: Zhang S, Tan HC, Ooi EE.
Journal: J Vis Exp. (2011)

Fluorescent "Turn-on" system utilizing a quencher-conjugated peptide for specific protein labeling of living cells
Authors: Arai S, Yoon SI, Murata A, Takabayashi M, Wu X, Lu Y, Takeoka S, Ozaki M.
Journal: Biochem Biophys Res Commun (2011): 211

Neuroanatomical basis of clinical joint application of "Jinggu" (BL 64, a source-acupoint) and "Dazhong" (KI 4, a Luo-acupoint) in the rat: a double-labeling study of cholera toxin subunit B conjugated with Alexa Fluor 488 and 594
Authors: Cui JJ, Zhu XL, Ji CF, Jing XH, Bai WZ.
Journal: Zhen Ci Yan Jiu (2011): 262

Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers
Authors: Kuwayama M, Shigemoto N, Oohara S, Tanizawa Y, Yamada H, Takeda Y, Matsuo T, Fukuda S.
Journal: J Microbiol Methods (2011): 119

Page updated on November 21, 2024

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Spectral properties

Correction Factor (260 nm)	0.21
Correction Factor (280 nm)	0.11
Extinction coefficient (cm ^-1 M ^-1)	75000¹
Excitation (nm)	491
Emission (nm)	516
Quantum yield	0.9¹