Clean-up of Antibody Labeling Reactions Using Amicon® Ultra Filters

Using a standard Amicon® Ultra centrifugal filter, a few rounds of diafiltration can efficiently remove unincorporated label from an antibody labeling reaction without diluting the sample. Use the protocol below to remove unreacted fluorescent labels using centrifugal ultrafiltration devices.

Removal of unreacted fluorescent label from antibody conjugation reaction

  1. Load 1 mg of fluorescent-labeled antibody solution in 2 mL into two Amicon® Ultra-4 mL 10 kDa MWCO devices and centrifuge at 4,000 x g for 10 minutes.
  2. Redilute retentates (about 50 μL each) to 2 mL with water and centrifuge again. Repeat this step twice.
  3. After each centrifugation, save samples of the retentate and filtrate for SDS-PAGE analysis and quantitation using UV-Vis spectroscopy.


SDS-PAGE gel of FITC-labeled BSA before and after each of four diafiltration cycles

Figure 1 shows the SDS-PAGE gel of FITC-labeled BSA before and after each of four diafiltration cycles. Unincorporated FITC is visible in the starting material and in the first filtrate. The majority of the free label is removed following one ultrafiltration cycle; free FITC is virtually undetectable after subsequent filtration cycles. Fluorescence measurement data in Figure 2 confirm this result. The free FITC signal is reduced by ~ 80% after the first ultrafiltration, and further reduced to 98% removal after subsequent filtration cycles. This demonstrates the viability of ultrafiltration as an alternative to gel filtration for clean-up of protein labeling reactions.