ReadiLink™ Rapid iFluor™ 647 Antibody Labeling Kit *Microscale Optimized for Labeling 50 µg Antibody Per Reaction*

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HeLa cells were incubated with mouse anti-tubulin followed by AAT’s iFluor™ 647 goat anti-mouse IgG conjugate (Red, Left) or Alexa Fluor<sup>®</sup> 647 goat anti-mouse IgG<sup>  </sup>(Red, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue, Cat#17530).
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Unit Size: Cat No: Price (USD): Qty:
2 Labelings 1235 $145

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Ex/Em (nm)654/674
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Category Superior Labeling Dyes
iFluor Dyes and Kits
Related General proteins
Labeling via Amino Groups
Secondary Reagents
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). iFluor™ 555 dyes have fluorescence excitation and emission maxima of ~550 nm and ~570 nm respectively. iFluor™ 647 family has the spectral properties essentially identical to those of Cy5® (Cy5® is the trademark of GE Healthcare). Compared to Cy5 probes iFluor™ 647 family has much stronger fluorescence and higher photostability. Their fluorescence is pH-independent from pH 3 to 11. These spectral characteristics make this new dye family a superior alternative to Cy5®. iFluor™ 647 family has become an excellent replacement for Cy5 and Alexa Fluor® 647 labeling dye (Alexa Fluor® is the trademark of Invitrogen). ReadiLink™ labeling kits essentially only require 2 simple mixing steps without a column purification needed. iFluor™ 647 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™ 647 dye provided in the kit is optimized for labeling ~50 µg antibody. iFluor™ 647 SE protein labeling kit provides a convenient method to label monoclonal, polyclonal antibodies or other proteins (>10 kDa) with the iFluor™ 647 SE.

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This protocol only provides a guideline, and should be modified according to your specific needs.
  1. Prepare protein solution (Solution A):
    For labeling 50 ug 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 uL of the target protein solution.
    Note 1: If you have a difference protein concentration, adjust the protein volume accordingly to make ~50 µg protein available for your labeling reaction.
    Note 2: For labeling 100 ug protein (assuming the target protein concentration is 1 mg/mL), mix 10 uL (10% of the total reaction volume) of Reaction Buffer (Component B) with 100 uL of the target protein solution.
    Note 3: 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 4: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.
    Note 5: The conjugation efficiency is significantly reduced if the protein concentration is less than 1 mg/mL. For optimal labeling efficiency the final protein concentration range of 1-2 mg/mL is recommended.

  2. Run conjugation reaction::
    1. Add the protein 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: Use both vials (Component A) of labeling dye to label 100 ug protein by dividing the 100 ug protein into 2x50 ug protein and reacting each 50 ug protein with one vial of labeling dye. Combine two 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.

  3. Stop Conjugation reaction:
    1. Add 5 uL (for 50 ug protein) or 10 uL (for 100 ug protein) which is 10% of the total reaction volume of TQ-Dyed Quench Buffer (Component C) into the conjugation reaction mixture (from step 2.2), mix them well.
    2. Incubate at room temperature for 10 minutes.
    3. The labeled protein (antibody) is now ready to use.

References & Citations

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

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

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

Additional Documents

Safety Data Sheet (SDS)

1. Fluorescent Labeling Probes & Kits

Application Notes
1. AssayWise Letters 2013, Vol 2(1)
2. AssayWise Letters 2012, Vol 1(1)

Certificate of Analysis