α1-2,4,6 Fucosidase O Digestion of Released Labeled Glycans Protocol
1. Digest 20 µg sample with PNGase F to release N-glycans
Note: NEB recommends deglycosylation with PNGase F (Glycerol Free), Recombinant (NEB #P0709) or Rapid PNGase F (NEB #P0710). See individual product pages for recommended digestion protocol.
2. Label released N-glycans by reductive amination (2AB, 2AA or Procainamide) or with an Instant Label (Instant 2AB, Instant Procainamide or RapiFluor) following manufacturer’s instructions.
3. Purify labelled glycan sample using a HILIC column or follow manufacturer’s instructions.
4. Lyophilize or dry (via speedvac) the purified, labelled sample.
5. Reconstitute dried sample in 9 µl water, and add 1 µl of 10X Glycobuffer 1 to create a 10 µl total reaction volume.
6. Add 2µl of α1-2,4,6 Fucosidase O (P0749)
Note: α1-2,4,6 Fucosidase O can be combined in a simultaneous digest with other exoglycosidases such as α2-3,6,8,9 Neuraminidase A (NEB# P0722), α1-3,4,6 Galactosidase (NEB# P0747), β1-4 Galactosidase S (NEB# P0745) and β-N-Acetylglucosaminidase S (NEB# P0744)
7. Incubate at 37°C for 3-18 hours for samples labelled by reductive amination or 18 hours for samples with Instant labels.
Note: NEB recommends deglycosylation with PNGase F (Glycerol Free), Recombinant (NEB #P0709) or Rapid PNGase F (NEB #P0710). See individual product pages for recommended digestion protocol.
2. Label released N-glycans by reductive amination (2AB, 2AA or Procainamide) or with an Instant Label (Instant 2AB, Instant Procainamide or RapiFluor) following manufacturer’s instructions.
3. Purify labelled glycan sample using a HILIC column or follow manufacturer’s instructions.
4. Lyophilize or dry (via speedvac) the purified, labelled sample.
5. Reconstitute dried sample in 9 µl water, and add 1 µl of 10X Glycobuffer 1 to create a 10 µl total reaction volume.
6. Add 2µl of α1-2,4,6 Fucosidase O (P0749)
Note: α1-2,4,6 Fucosidase O can be combined in a simultaneous digest with other exoglycosidases such as α2-3,6,8,9 Neuraminidase A (NEB# P0722), α1-3,4,6 Galactosidase (NEB# P0747), β1-4 Galactosidase S (NEB# P0745) and β-N-Acetylglucosaminidase S (NEB# P0744)
7. Incubate at 37°C for 3-18 hours for samples labelled by reductive amination or 18 hours for samples with Instant labels.
