FAQ: How do I eliminate Endo D from a reaction?

Endo D can be removed from a deglycosylation reaction using NEB’s Magnetic Chitin Beads (NEB #E8036). Typical reaction conditions for removing 1-5 µL of Remove-iT® Endo D using magnetic chitin beads are as follows: Materials: Endo D (NEB #P0742), Chitin Magnetic Beads (NEB #E8036), Magnetic Separation Rack (NEB #S1506, 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.
  2. With the eppendorf on the magnetic separation rack, wash the magnetic chitin beads 2 x 500 µl with 50 mM NH4Formate 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 NH4Formate pH 4.4 (or buffer of choice). Pipette of the supernatant from each wash and keep.
  7. Combine all supernatants from steps 5 & 6, as these are 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.