This and the below procedures were developed around labeling 50 μg of IgG but are compatible with scaling up or down. In our case, normal Rabbit IgG (ThermoFisher Cat: 31235) is combined with DBCO-Peg5-NHS Ester (linker; 10 mM in DMSO; Click Chemistry Tools Cat: 1378531-80-6) in 20 molar excess (50 μg of Rabbit IgG and 4.6 μg of linker). This is brought to a volume of 100μl with PBS and allowed to incubate for 30 minutes at 25°C. After incubation, the solution is added to an Amicon Ultra 0.5ml 100 kDa centrifugal filter (Fisher Scientific Cat: UFC5100BK) and spun for 5 minutes at 14,000 × g. The filter is then placed into a new tube, and PBS is added to the top of the filter in order to bring the total volume back to 100 μl and is spun again for 5 minutes at 14,000 × g. This wash step is repeated twice more (three total). Finally, the filter is flipped upside down and placed in a clean tube and spun for 1 minute at 1000 × g to collect the retentate. The retentate absorbance is measured at 309nm, where the linker strongly absorbs, and 280nm, where the antibody strongly absorbs, using a NanoDrop spectrophotometer (Thermo Scientific).
Amicon ultra 0.5ml 100 kda centrifugal filter
Manufactured by Thermo Fisher Scientific
The Amicon Ultra 0.5ml 100 kDa centrifugal filter is a laboratory device designed for sample concentration and buffer exchange. It features a 100 kDa molecular weight cut-off membrane that allows the separation of molecules based on their size during centrifugation.
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2 protocols using amicon ultra 0.5ml 100 kda centrifugal filter
1
IgG Labeling with DBCO-Peg5-NHS Ester
2
Antibody Labeling with DBCO-Peg5-NHS Ester
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This and the below procedures were developed around labeling 50 mg of IgG but is compatible with scaling up or down. In our case, normal Rabbit IgG (ThermoFisher Cat: 31235) is combined with DBCO-Peg5-NHS Ester (linker; 10 mM in DMSO; Click Chemistry Tools Cat: 1378531-80-6) in 60 molar excess (50 mg of Rabbit IgG and 4.6 mg of linker). This is brought to a volume of 100ml with PBS and allowed to incubate for 30 minutes at 25°C. After incubation, the solution is added to an Amicon Ultra 0.5ml 100 kDa centrifugal filter (Fisher Scientific Cat: UFC5100BK) and spun for 5 minutes at 14,000 x g. The filter is then placed into a new tube and PBS is added to the top of the filter in order to bring the total volume back to 100 ml and is spun again for 5 minutes at 14,000 x g. This wash step is repeated twice (three total). Finally, the filter is flipped upside down and placed in a clean tube and spun for 1 minute at 1000 x g to collect the retentate. The retentate absorbance is measured at 309nm, where the linker strongly absorbs, and 280nm, where the antibody strongly absorbs, using a NanoDrop spectrophotometer (Thermo Scientific).
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