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
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.
Figure 2.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.