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 1.0M DTT stock per 100 µL of antibody solution and mix well. Let the antibody solution stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).

Purify the reduced antibody over a desalting column pre-equilibrated with 50 mM MES Buffer (pH=6.0-6.5) with 2 mM EDTA.

Measure the antibody concentration with Nanodrop. (Con. (mg/ml) = A280nm/1.4).
Note: The reduced antibody is not stable; the conjugation reaction needs to be carried out the conjugation as soon as possible after purification.

Reconstitute PerCP and PerCP-Tandem in 100 µL ddH2O to 10 mg/ml.
Note: Reconstituted PerCP and PerCP-Tandem can be stored at 4 °C for a month, please kept it from light. If PerCP and PerCP-Tandem is already in liquid format, reconstitution is not needed.

Prepare Sulfo-SMCC or SMCC stock solution in DMSO to 10 mg/ml. Add 5~10.0 µL 10 mg/ml stock solution per 1mg PerCP and PerCP-Tandem and mix well.

Purify the activated PerCP and PerCP-Tandem over a desalting column pre-equilibrated with 50 mM MES Buffer (pH=6.0-6.5) with 2 mM EDTA.

Measure the PerCP and PerCP-Tandem concentration with Nanodrop.

Mix reduced antibody with pre-activated PerCP and PerCP-Tandem directly to at the ratio of 100 µg PerCP and PerCP-Tandem / 100µg reduced antibody. (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.)

Reaction for 60-120 min 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.