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Nevada Proteomics Center

Phone(775) 784-6337: 2-D Gels
Phone(775) 784-1590: Mass Spectrometry
Fax(775) 784-1419
Emailquilici@unr.edu
Location School of Medicine
Manville Medical Building, Rooms 15-17, MS 0330
Address 1664 N. Virginia Street
Reno,  NV  89557-MS 0330
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Protocol: Use of PNGase F and Other Glycosidases

PNGase F Sources:

  1. New England BioLabs, catalog no. P0704 or P0705
  2. Sigma, catalog no. P7367
  3. Promega, catalog no. V4831
  4. QA Bio, catalog no. E-PNG01• 5% SDS
  5. Bio-Rad, catalog no. 170-6883

Protocol from Promega:

  • 5% SDS
  • 1M DTT
  • 0.5M sodium phosphate buffer (pH 7.5)
  • 10% NP-40
  1. 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.
  2. Add 1µl 5% SDS.
  3. Add 1µl of 1M DTT.
  4. Denature sample by heating at 95°C for 5 minutes.
  5. Cool sample for 5 minutes at room temperature.
  6. 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.
  7. Add 2µl of 10% NP-40.
    Note: Triton® X-100 may be substituted for NP-40.
  8. Add 2µl of recombinant PNGase F.
  9. 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
  • β-N-Acetylglucosaminidase
  • 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))
  • Endoglycosidases

Glycosidase Kits

QA Bio has a number of kits that will remove glycans from proteins.

DeGlycoMx

QA Bio Catalog No.: KE-DGMX or KE-DG01

Completely removes all N-linked glycans and most O-linked glycans.

Contains:

  • PNGase F
  • Sialidase
  • ß-Galactosidase
  • Glucosaminidase
  • O-Glycosidase

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.

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University of Nevada, Reno

University of Nevada, Reno
1664 N. Virginia Street
Reno,  NV  89557-

(775) 784-1110
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