Amicon ultra 0.5 centrifugal filtration units

To a suspension of 150 μg of Fab_ns in PBS pH 7.4 (14.64 mg/mL) was added 5.99 μL of a fresh TCEP solution (10 mM in water, 20 eq.). The reaction mixture was stirred on a thermomixer (37 °C) for 2 hours. The reductant was then removed using centrifugal filtration units with a 3,000 Da molecular weight cut off (AmiconTM Ultra 0.5 Centrifugal Filtration Units, Millipore Corp. Billerica, MA) at 4 °C into fresh PBS pH = 7.4. 4.50 μL of a DiPODS-FITC solution (10 mM in DMSO) was immediately added to the reduced Fab_ns. The reaction mixture was stirred on a thermomixer (37 °C) for 16 hours. The conjugate was then purified on a size exclusion column (Sephadex G-25 M, PD-10 column, GE Healthcare; dead volume = 2.5 mL, eluted with 2 mL of PBS, pH 7.4) and concentrated using centrifugal filtration units with a 30,000 Da molecular weight cut off (Amicon™ Ultra 0.5 Centrifugal Filtration Units, Millipore Corp. Billerica, MA).

To a suspension of 150 μg of Fab_HER2 in PBS pH 7.4 (1.19 mg/mL) was added 5.99 μL of a fresh TCEP solution (10 mM in water, 20 eq.).The reaction mixture was stirred on a thermomixer (37 °C) for 2 hours. The reductant was then removed using centrifugal filtration units with a 3,000 Da molecular weight cut off (AmiconTM Ultra 0.5 Centrifugal Filtration Units, Millipore Corp. Billerica, MA) at 4 °C into fresh PBS pH = 7.4. The appropriate volume of a DiPODS-FITC solution (10 mM in DMSO) was immediately added to the reduced Fab_HER2. The reaction mixture was stirred on a thermomixer (25 °C or 37 °C) for 2, 4, 8, 16, and 24 h. The conjugate was then purified on a size exclusion column (Sephadex G-25 M, PD-10 column, GE Healthcare; dead volume = 2.5 mL, eluted with 2 mL of PBS, pH 7.4) and concentrated using centrifugal filtration units with a 30,000 Da molecular weight cut off (Amicon™ Ultra 0.5 Centrifugal Filtration Units, Millipore Corp. Billerica, MA).

To a suspension of 150 μg of Fab_ns in PBS pH 7.4 (14.64 mg/mL) was added 5.99 μL of a fresh TCEP solution (10 mM in water). The reaction mixture was stirred on a thermomixer (25 °C or 37 °C) for 2 hours. The reductant was then removed using centrifugal filtration units with a 3,000 Da molecular weight cut off (Amicon™ Ultra 0.5 Centrifugal Filtration Units, Millipore Corp. Billerica, MA) at 4 °C into fresh PBS pH = 7.4. The reaction mixture was immediately stored at 4 °C.