Home Protocols Rapid PNGase F Protocols (P0710)

Rapid™ PNGase F Protocols (P0710)

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, are suitable for incubation.

One-step Protocol:
  1. Combine up to 100 μg of antibody and H2O to a volume of 16 μl.
  2. Add 4 μl of Rapid PNGase F Buffer (5X) to make a 20 μl total reaction volume.
  3. Add 1 μl of Rapid PNGase F.
  4. Incubate 10 minutes at 50°C.
  5. Prepare N-glycans for derivatization (i.e. reductive amination) for downstream analysis. To prepare a deglycosylated protein for mass spectrometry analysis, exchange the buffer by micro dialysis or micro filtration. Refer to the “Glycoproteomics: Buffer Exchange Protocols” for more detail. 
Two-step Protocol:

Some antibodies (i.e. Fab N-glycans) require a preheating step for efficient deglycosylation.
  1. Combine up to 100 μg of antibody and H2O to a volume of 16 μl.
  2. Add 4 μl of Rapid PNGase F Buffer (5X) to make a 20 μl total reaction volume.
  3. Incubate at 80°C for 2 minutes, cool down.
  4. Add 1 μl of Rapid PNGase F.
  5. Incubate 10 minutes at 50°C.
  6. Prepare N-glycans for derivatization (i.e. reductive amination) for downstream analysis. To prepare a deglycosylated protein for mass spectrometry analysis, exchange the buffer by micro dialysis or micro filtration. Refer to the “Glycoproteomics: Buffer Exchange Protocols” for more detail. 
Notes:

  • The target protein should be in a solution compatible with Rapid PNGase F activity. Avoid buffers containing SDS, as it inhibits PNGase F. Common stabilizing reagents such as Tween, Triton X-100, NP-40, octyl glucoside and non-detergent sulfobetaine, as well as traces of organic solvents, can prevent optimal rapid deglycosylation. 
  • To prepare samples for liquid chromatography and/or mass spectrometry, N-glycans can be isolated by conventional solid phase extraction methods such as C18 and graphitized carbon, refer to “Glycan SPE C18 and Graphitized Carbon Protocols”. 
  • Although this product has been optimized for the rapid removal of N-glycans from antibodies, it can be utilized with various other glycoproteins.