Home FAQs What are the typical reaction conditions for Rapid PNGase F?

FAQ: What are the typical reaction conditions for Rapid PNGase F?

For optimal heat transfer, use 0.2ml 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. Reactions may be scaled-up linearly to accommodate larger amounts of glycoprotein and/or larger reaction volumes.

One step protocol:
  1. Combine up to 100 μg of antibody and H2O to a volume of 16 μl.
  2. Add 4 μl of 5X Rapid PNGase F Buffer 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, buffer exchange 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 pre-heating 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 5X Rapid PNGase F Buffer 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, buffer exchange by micro-dialysis or micro-filtration. Refer to the “Glycoproteomics: Buffer Exchange Protocols” for more detail.