Protocol: Use of PNGase F and Other Glycosidases
PNGase F Sources:
- New England BioLabs, catalog no. P0704 or P0705
- Sigma, catalog no. P7367
- Promega, catalog no. V4831
- QA Bio, catalog no. E-PNG01• 5% SDS
- Bio-Rad, catalog no. 170-6883
Protocol from Promega:
- 5% SDS
- 1M DTT
- 0.5M sodium phosphate buffer (pH 7.5)
- 10% NP-40
- Add up to 50µg of the target glycoprotein in water (or a compatible buffer at a low
ionic strength) to a final volume of 12µl.
- Add 1µl 5% SDS.
- Add 1µl of 1M DTT.
- Denature sample by heating at 95°C for 5 minutes.
- Cool sample for 5 minutes at room temperature.
- Add 2µl of 0.5M sodium phosphate buffer (pH 7.5).
Note: Other buffers can be used, if they are within the acceptable pH range for
PNGase F, pH 6-10.
- Add 2µl of 10% NP-40.
Note: Triton® X-100 may be substituted for NP-40.
- Add 2µl of recombinant PNGase F.
- Incubate at 37°C for 1-3 hours.
Note: PNGase F cannot cleave when an α1-3 Fucose is attached to the core GlcNAc.
Other Commercially Available Glycosidases
New England BioLabs has a large variety of glycosidases for use in removing or characterizing glycans.
- β1-3 Galactosidase
- β1-4 Galactosidase
- α1-2,3 Mannosidase
- α1-6 Mannosidase
- α1-3,6 Galactosidase
- α1-2 Fucosidase
- O-Glycosidase (Endo-α-N-Acetylgalactosaminidase)
- Neuraminidase (acetyl-neuraminyl hydrolase (sialidase))
QA Bio has a number of kits that will remove glycans from proteins.
QA Bio Catalog No.: KE-DGMX or KE-DG01
Completely removes all N-linked glycans and most O-linked glycans.
- PNGase F
Orela O-linked Glycan Release Kit
QA Bio Catalog No.: LL-ORELA-A2
Chemically releases O-glycans, then fluorescently labels the Ser or Thr to which the glycans were attached.
Endo F Multi-Kit
QA Bio Catalog No.: KE-EFX3)
Cleaves after the N-acetyl glucosamine attached to Asn on a variety of N-linked glycans. A GlcNAc remains attached at the location of polysaccharide chain.