Remove-iT® PNGase F (NEB #P0706)
Chitin Magnetic Beads (NEB #E8036)
Magnetic Separation Rack (NEB #S1506 or NEB #S1509)
- Pipette 50 µl Chitin Magnetic Beads into an eppendorf tube and place the eppendorf in a magnetic separation rack. Let the magnet attract the chitin beads, then pipette off the liquid supernatant and discard.
- With the eppendorf on the magnetic separation rack, wash the magnetic chitin beads 2 x 500 µl with 50 mM NH4 Formate pH 4.4 (or buffer of choice). Pipette of the supernatant and discard.
- Add the deglycosylated glycoprotein sample into the eppendorf with magnetic chitin beads.
- Rock the deglycosylated glycoprotein sample with the magnetic chitin beads for 10 minutes at 4°C.
- Place the eppendorf back on the magnetic separation rack, and allow the magnet to attract the chitin beads. Pipette off the supernatant and keep.
- Wash the magnetic chitin beads 3 x 100 µl with 50 mM NH4 Formate pH 4.4 (or buffer of choice). Pipette of the supernatant from each wash and keep.
- Combine all supernatants from steps 5 & 6, as these are the deglycosylated glycoprotein.
- Analyze sample by method of choice.
Note - Removal of Remove-iT® PNGase F from the deglycosylation reaction can be scaled up linearly with larger magnetic chitin bead volumes.