ReadiLink™ Rapid iFluor® 633 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction

AAT Bioquest's iFluor® dyes are developed for labeling proteins, in particular, antibodies. These dyes are optimized to have minimal fluorescence quenching effect on proteins and nucleic acids. Our iFluor® 633 dyes have fluorescence excitation and emission maxima close to ~633 nm and ~655 nm respectively with good photostability. Our in-house comparable studies indicated that our iFluor® 633 dyes are significantly brighter than the corresponding Cy5®. These spectral characteristics make them a superior alternative to Cy5® and Alexa Fluor®633 (Cy5® and Alexa Fluor® are the trademarks of GE Healthcare and Invitrogen respectively). iFluor® 633 conjugates have been widely used in fluorescence animal imaging applications. ReadiLink™ labeling kits essentially only require 2 simple mixing steps without a column purification needed. iFluor® 633 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® 633 dye provided in the kit is optimized for labeling ~50 µg antibody. iFluor® 633 SE protein labeling kit provides a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the iFluor® 633 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® 350 Antibody Labeling Kit Production Scale	345	450	20000¹	0.95¹	0.83	0.23
ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	491	516	75000¹	0.9¹	0.21	0.11
ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit Production Scale	491	516	75000¹	0.9¹	0.21	0.11
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® 555 Antibody Labeling Kit Production Scale	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® 594 Antibody Labeling Kit Production Scale	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® 647 Antibody Labeling Kit Production Scale	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® 750 Antibody Labeling Kit Production Scale	757	779	275000¹	0.12¹	0.044	0.039
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
Show More (6)

Citations

View all 5 citations: Citation Explorer

Vnn1 pantetheinase limits the Warburg effect and sarcoma growth by rescuing mitochondrial activity
Authors: Giessner, Caroline and Millet, Virginie and Mostert, Konrad J and Gensollen, Thomas and Manh, Thien-Phong Vu and Garibal, Marc and Dieme, Binta and Attaf-Bouabdallah, Noudjoud and Chasson, Lionel and Brouilly, Nicolas and others, undefined
Journal: Life Science Alliance (2018): e201800073

A novel CD81 homolog identified in lamprey, Lampetra japonica, with roles in the immune response of lamprey VLRB+ lymphocytes
Authors: Liang, Wenjing and Gao, Miceng and Song, Xueying and Han, Yinglun and Go, Meng and Su, Peng and Li, Qingwei and Liu, Xin
Journal: Acta biochimica et biophysica Sinica (2018)

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

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Spectral properties

Correction Factor (260 nm)	0.062
Correction Factor (280 nm)	0.044
Extinction coefficient (cm ^-1 M ^-1)	250000¹
Excitation (nm)	640
Emission (nm)	654
Quantum yield	0.29¹