Endo D Removal Magnetic Chitin Bead Protocol (P0742)

Endo D (NEB #P0742)
Chitin Magnetic Beads (NEB #E8036)
Magnetic Separation Rack (NEB #S1506 or NEB #S1509)
  1. 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 the supernatant.
  2. 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.
  3. Add the deglycosylated glycoprotein sample into the eppendorf with magnetic chitin beads.
  4. Rock the deglycosylated glycoprotein sample with the magnetic chitin beads for 10 minutes at 4°C.
  5. Place the eppendorf back on the magnetic separation rack, and allow the magnet to attract the chitin beads. Pipette off the supernatant and keep.
  6. Wash the magnetic chitin beads 3 x 100 μl with 50 mM NH4 Formate, pH 4.4 (or buffer of choice). Pipette off the supernatant from each wash and keep.
  7. Combine all supernatants from steps 5 & 6, as these contain the deglycosylated glycoprotein.
  8. Analyze sample by method of choice
Notes: Removal of Endo D from the deglycosylation reaction can be scaled up linearly with larger magnetic chitin bead volumes. The ideal reaction volume for 50 μl of chitin resin is in the range of equal volume to no more than 5X bead bed volume.