Remove-iT® Endo D (NEB #P0742)
Chitin Magnetic Beads (NEB #E8036)
Magnetic Separation Rack (NEB #S1506 or
- 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.
- 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
- Place the eppendorf back on the magnetic
separation rack, and allow the magnet to attract
the chitin beads. Pipette off the supernatant
- 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.
- Combine all supernatants from steps 5 & 6, as
these contain the deglycosylated glycoprotein.
- Analyze sample by method of choice
Notes: Removal of Remove-iT® 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.