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.
- 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).
- 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).
- 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
- 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).
||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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