This protocol is intended only as a general guide; please adapt it to your specific needs.
1 Protein Preparation
(1) For optimal labeling, prepare the protein (antibody) at 2mg/mL.
(2)Adjust the protein solution to pH 8.5 ± 0.5. If the pH is below 8.0, use 1M sodium bicarbonate to raise it.
(3) Labeling efficiency drops sharply when the protein concentration is < 2mg/mL. For best results, work within a final protein concentration range of 2–10mg/mL.
(4) The protein must be in a buffer free of primary amines (e.g., Tris or glycine) and ammonium ions, as these will reduce labeling efficiency.
2 Dye Preparation (using AQC as an example)
(1) Add anhydrous DMSO to the AQC vial to prepare a 10mM stock solution. Mix thoroughly by pipetting or vortexing.
3 Calculating Dye Amount
(1) The amount of AQC required depends on the quantity of protein to be labeled. The optimal molar ratio of AQC to protein is ~10:1.
(2) Example: To label 500µL of 2mg/mL IgG (MW = 150,000) with AQC dissolved in 100µL DMSO (1mg AQC), the required volume of AQC is 1.9µL.
Detailed calculation:
mmol(IgG) = [mg/mL(IgG) × mL(IgG)] / MW(IgG)= (2mg/mL × 0.5mL) / 150,000 mg/mmol= 6.7 × 10⁻⁶mmol
mmol(AQC) = mmol(IgG) × 10= 6.7 × 10⁻⁶mmol × 10= 6.7 × 10⁻⁵mmol
µL(AQC) = [mmol(AQC) × MW(AQC)] / (mg/µL AQC)= (6.7 × 10⁻⁵mmol × 285.25mg/mmol) / 0.01mg/µL≈ 1.9µL AQC
(4) Running the Conjugation Reaction
(1) Slowly add the freshly prepared 10mM AQC stock to 0.5mL of protein solution while gently swirling. Briefly centrifuge to collect the sample at the bottom of the tube. Do not vortex to avoid denaturation or loss of activity.
(2) Incubate the reaction tube at room temperature in the dark, gently swirling every 10–15min, for a total of 60min.
5 Purifying the Conjugate (example using a Sephadex G-25 column)
(1) Equilibrate a Sephadex G-25 column according to the manufacturer’s instructions.
(2) Load the reaction mixture onto the top of the column bed.
(3) As soon as the sample has entered the resin, immediately add PBS (pH 7.2–7.4).
(4) Continue adding PBS (pH 7.2–7.4) until the desired fractions have been collected and the column run is complete.
References:
[1]Bosch L, Alegría A, Farré R. Application of the 6-aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC) reagent to the RP-HPLC determination of amino acids in infant foods. J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Feb 2;831(1-2):176-83.
