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

Indirect immunofluorescence staining of tubulin. HeLa cells were stained with mouse anti-tubulin followed by a goat anti-mouse IgG labeled using the ReadiLink™ Rapid iFluor® 633 Antibody Labeling Kit (Cat No. 1260). Cell nuclei were counterstained using DAPI (Cat No. 17507).
Indirect immunofluorescence staining of tubulin. HeLa cells were stained with mouse anti-tubulin followed by a goat anti-mouse IgG labeled using the ReadiLink™ Rapid iFluor® 633 Antibody Labeling Kit (Cat No. 1260). Cell nuclei were counterstained using DAPI (Cat No. 17507).
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Catalog Number1260
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
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 yield0.291
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


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
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.

 

readilinkworkflow

 

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


Component A: iFluor™ 6332 vials (One vial is for 50 μg protein)
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, 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
  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


Open in Advanced Spectrum Viewer
spectrum

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 yield0.291

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
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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
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Neuroanatomical characteristics of acupoint "Chengshan" (BL 57) in the rat: a cholera toxin subunit B conjugated with Alexa Fluor 488 method study
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Novel Alexa Fluor-488 labeled antagonist of the A(2A) adenosine receptor: Application to a fluorescence polarization-based receptor binding assay
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