Conjugation Protocol for Amine Reactive Dyes

This is intended as a guide only for protein/antibody labeling; optimal conjugation protocols may vary depending on the target being labeled.

Reagents

  • Prepare a 10 mM stock solution of NHS ester dye in anhydrous DMSO or DMF. Briefly vortex.
  • Prepare protein/antibody to be conjugated at approximately 3.0 mg/mL (minimum recommended protein concentration is 2.0 mg/mL) in either sodium borate (50 mM, pH 8.5) or carbonate buffer (100 mM, pH 8-8.5). N.B. for carbonate buffer, we recommend adding 75 mg/mL Sodium Bicarbonate solution as indicated below to adjust the pH of the starting protein/antibody PBS solution to approximately 8.25. Protein/Antibody Volume (µL) x 0.112 = Volume Sodium Bicarbonate required (µL).

Conjugation

  • Add dye solution to protein solution at the appropriate molar ratio (see note above) while stirring/gently vortexing.
  • Incubate at room temperature for 60 minutes in the dark.
  • Quench the reaction by adding Tris-HCl or Glycine (pH 7.4, 50 - 100 mM final concentration), incubate with stirring for 10-15 minutes at room temperature (this step is optional).

Purification

  • Remove excess dye with a Zeba™ Spin desalting column (Thermo), with appropriate MWCO, or a PD MiniTrap™ G-25 (GE Healthcare), following the manufacturer’s instructions.

Degree of Labeling Calculation

  • Dilute the protein-dye conjugate to approximately 0.1 mg/mL and measure the absorbance at 280 nm (protein A280) and at the maximum absorbance wavelength (Amax) for the fluorescent dye used (please refer to the individual product descriptions for the max. λabs values).
  • Calculate the corrected A280 (A280C) using the following equation:

    A280c = A280 - (Amax x CF)     

    Please refer to the individual product descriptions for the correction factor (CF) values for the fluorescent dye used.

  • Calculate the final protein concentration ([protein] in mg/mL) using the corrected A280C value calculated above, the extinction coefficient (ε) for your protein and the Beer-Lambert Law equation

    A280 = ε x [protein] x l

  • Calculate the final F:P ratio (Degree of Labeling), where εdye is the extinction coefficient for the fluorescent dye used (please refer to the individual product descriptions for this value).
    F:P = Amax x MWprotein
    [protein] x εdye

Zeba is a trademark of Thermo Scientific and MiniTrap is a trademark of GE Healthcare.

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