ReadiLink™ Rapid FITC Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction

HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by AAT’s FITC goat anti-mouse IgG conjugate (Green, Left) or Jackson’s FITC goat anti-mouse IgG conjugate (Green, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat# 17530).

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Spectral properties

Correction Factor (280 nm)	0.35
Extinction coefficient (cm ^-1 M ^-1)	73000
Excitation (nm)	491
Emission (nm)	516
Quantum yield	0.92

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

Correction Factor (280 nm)

0.35

Extinction coefficient (cm ^-1 M ^-1)

73000

Excitation (nm)

491

Emission (nm)

516

Quantum yield

0.92

FITC is still the most popular fluorescent labeling dye for preparing green fluorescent bioconjugates. However, most of commercial FITC labeling kits require intensive hands-on time. This FITC ReadiLink™ labeling kit essentially only requires 2 simple mixing steps without a column purification needed. The kit provides all the essential components for labeling ~2x50 ug antibody. Each of the two vials of FITC dye provided in the kit is optimized for labeling ~50 µg antibody. This FITC protein labeling kit provides a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa).

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.

Components

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

Open in Advanced Spectrum Viewer

Spectral properties

Correction Factor (280 nm)	0.35
Extinction coefficient (cm ^-1 M ^-1)	73000
Excitation (nm)	491
Emission (nm)	516
Quantum yield	0.92

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield
ReadiLink™ Rapid Cy3 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	555	569	150000¹	0.15¹
ReadiLink™ Rapid Cy5 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	651	670	250000¹	0.27¹, 0.4²
ReadiLink™ Rapid Cy7 Antibody Labeling Kit Microscale Optimized for Labeling 50 μg Antibody Per Reaction	756	779	250000	0.3
ReadiLink™ Rapid XFD488 Antibody Labeling Kit XFD488 Same Structure to Alexa Fluor™ 488	499	520	73000	0.92¹
ReadiLink™ Rapid XFD555 Antibody Labeling Kit XFD555 Same Structure to Alexa Fluor™ 555	553	568	155000	0.1¹
ReadiLink™ Rapid XFD594 Antibody Labeling Kit XFD594 Same Structure to Alexa Fluor™ 594	590	618	92000	0.66¹
ReadiLink™ Rapid XFD647 Antibody Labeling Kit XFD647 Same Structure to Alexa Fluor™ 647	650	671	270000	0.33¹
ReadiLink™ Rapid XFD750 Antibody Labeling Kit XFD750 Same Structure to Alexa Fluor™ 750	752	776	290000	0.12¹

Images

Figure 1. HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by AAT’s FITC goat anti-mouse IgG conjugate (Green, Left) or Jackson’s FITC goat anti-mouse IgG conjugate (Green, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat# 17530).

Citations

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Utilizing the nanosecond pulse technique to improve antigen intracellular delivery and presentation to treat tongue squamous cell carcinoma
Authors: Gao, Cen and Zhang, Xingxing and Chen, Jian and Zhao, Jiayuan and Liu, Yanmei and Zhang, Jue and Wang, Jing
Journal: Medicina oral, patologia oral y cirugia bucal (2018): e344

Role of reactive oxygen species-mediated MAPK and NF-$\kappa$B activation in polygonatum cyrtonema lectin-induced apoptosis and autophagy in human lung adenocarcinoma A549 cells
Authors: Liu, Tao and Wu, Lei and Wang, Di and Wang, Haiyang and Chen, Jinwu and Yang, Chunlan and Bao, Jinku and Wu, Chuanfang
Journal: The Journal of Biochemistry (2016): 315--324

Determination of sodium benzoate in food products by fluorescence polarization immunoassay
Authors: Ren, Linlin and Meng, Meng and Wang, Peng and Xu, Zhihuan and Eremin, Sergei A and Zhao, Junhong and Yin, Yongmei and Xi, Rimo
Journal: Talanta (2014): 136--143

Complexes containing cationic and anionic pH-sensitive liposomes: comparative study of factors influencing plasmid DNA gene delivery to tumors
Authors: Chen, Yan and Sun, Ji and Lu, Ying and Tao, Chun and Huang, Jingbin and Zhang, He and Yu, Yuan and Zou, Hao and Gao, Jing and Zhong, Yanqiang
Journal: International journal of nanomedicine (2013): 1573

Swimming behavior of the monotrichous bacterium Pseudomonas fluorescens SBW25
Authors: Ping, Liyan and Birkenbeil, Jan and Monajembashi, Shamci
Journal: FEMS microbiology ecology (2013): 36--44

References

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