Conjugation of Antibodies to APC, PE, PerCP and Tandem Dyes

Below is an example protocol for conjugation of antibodies with APC, PE, PerCP or their respective tandem dyes. The protocol provides general guidelines; actual conjugation steps may require further optimization depending on the antibodies and labels used.

Conjugation Protocol

1. Reduction of Antibody

Prepare a fresh solution of 1.0 M DTT (15.4 mg/100 µl) in distilled water. Antibody solutions should be at 1 mg/ml or higher for best results. The reduction can be carried out in different buffers for example: MES, phosphate, and TRIS buffers (pH range 6 to 8). The antibody should be concentrated if less than 1 mg/ml.
Add 2 µL of 1.0 M DTT stock per 100 µL of antibody solution and mix well. Let the antibody solution stand at room temperature for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
Purify the reduced antibody over a desalting column (Catalog 60500) pre-equilibrated with 50 mM MES Buffer (pH = 6.0–6.5) with 2 mM EDTA.
Measure the antibody concentration with Nanodrop.
(Concentration (mg/ml) = A280 nm / 1.4)
Note: The reduced antibody is not stable; the conjugation reaction needs to be carried out as soon as possible after purification.

2. Activation of APC, PE, PerCP and Tandem Dyes

Reconstitute PerCP, APC, PE, or corresponding tandem dyes in 100 µL ddH₂O to 10 mg/mL.
Note: Reconstituted dyes can be stored at 4 °C for one month; protected from light. If the dye is already in liquid format, reconstitution is not needed.
Prepare Sulfo-SMCC or SMCC stock solution in DMSO.
• Add 5–10 µL of 10 mg/mL SMCC per 1 mg dye (PerCP, PerCP-Tandem)
• Add 2–6 µL of 10 mM SMCC per 1 mg dye (APC, APC-Tandem, PE, PE-Tandem)
Mix well.
Purify the activated dye over a desalting column pre-equilibrated with 50 mM MES Buffer (pH = 6.0–6.5) with 2 mM EDTA.
Measure the dye concentration with Nanodrop.

3. Reduced Antibody Conjugation with Activated Dye

Mix reduced antibody with pre-activated dye directly at the following ratios:
• 100 µg dye / 100 µg reduced antibody (PerCP / PerCP-Tandem)
• 130 µg dye / 100 µg reduced antibody (APC / APC-Tandem)
• 250 µg dye / 100 µg reduced antibody (PE / PE-Tandem)
Note: Keep the antibody concentration lower than 1.5 mg/ml; please add MES buffer to the reaction mixture if antibody concentration is higher than 1.5 mg/ml.)
React for 60–120 minutes at room temperature.
After the reaction, block the free sulfhydryls on the antibody. Prepare a fresh solution of 10 mg/mL NEM in DMSO; add 3.4 µL per mg of antibody and rotate for 20 minutes at room temperature.

4. Purification

Antibody/dye or antibody/tandem conjugates may be further purified through size-exclusion chromatography to obtain best performance.
Note: The conjugate solution is recommended to be stored at 2–8 °C and protected from light.

Document: 02.0246.260102r1

Last updated Fri Jan 02 2026