Home Protocols Rapid PNGase F (non-reducing format) (P0711) Reaction Protocol

Rapid PNGase F (non-reducing format) (P0711) Reaction Protocol

The quantity of enzyme recommended is sufficient for the deglycosylation of 10 μg of antibody under non-reducing conditions. The optimal amount of starting material will be determined by the nature of a sample (glycan diversity) and the particular downstream analysis that will be performed. Reactions may be scaled-up linearly to accommodate larger amounts of glycoprotein. Optimal incubation times may vary for a particular substrate.

For optimal heat transfer, use 0.2 ml thin wall microtubes or alternatively, 1.5 ml centrifuge tubes. A thermal cycler with heated lid, or a microtube heat block plate, are suitable for incubation.

1.    Combine 10 μg of antibody and H2O to a volume of 8 μl
2.    Add 2 μl of 5X Rapid PNGase F (non-reducing format) Buffer to make a 10 μl total reaction volume
3.    Incubate 5 minutes at 75°C
4.    Add 1 μl of Rapid PNGase F (non-reducing format)
5.    Incubate 10 minutes at 50°C
6.    Prepare antibody sample for SDS-PAGE or mass spectrometry analysis.


Notes:
1.    In many cases, the amount of antibody can be increased to 30 μg per 10 μl reaction. 1 µl of Rapid PNGase F (non-reducing format) in a 10 μl total reaction volume is optimal for rapid deglycosylation of up to 10 μg of antibodies containing both Fc and Fab glycosylation (such as Cetuximab), 15 μg of a mouse monoclonal antibody isotype IgG2a, and 30 μg of less complex antibodies, such as Rituximab. Reactions may be scaled-up linearly to accommodate larger amounts of antibody and/or glycoprotein.
2.    Some targets have a strict requirement for reducing agents to become fully deglycosylated (for instance: IgA1, Fetuin and chorionic gonadotropin). In this case, Rapid PNGase F (NEB #P0710) is recommended.
3.    The amount of Rapid PNGase F (non-reducing format) Buffer can be increased up to 4 μl to facilitate rapid deglycosylation of complex substrates.
4.    Although this product has been optimized for the rapid deglycosylation of antibodies, it can be utilized with various other glycoproteins (conditions will require optimization).