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Immobilized PNGASE F workflow

Rapid Removal of N-glycans

Immobilized PNGase F is a resin with the PNGase F enzyme covalently coupled to agarose beads in spin column format for easy removal of N-glycans on antibodies, fusion proteins and other N-glycosylated proteins.
 
Immobilized PNGase F is available in the following product formats:

  • Immobilized PNGase F- For removal of N-glycans from 0.2 mg glycoprotein under native conditions.
  • Immobilized PNGase F Denaturing - For removal of N-glycans from 0.2 mg glycoprotein under denaturing conditions. The MS compatible RapiGest™ SF Surfactant (see below) is included.

Immobilized PNGase F

Immobilized PNGase F is a resin with the PNGase F (Peptide N-glycosidase F) enzyme covalently coupled to agarose beads for removal of N-glycans on antibodies, fusion proteins and other N-glycosylated proteins. The enzyme is expressed in E. coli from a recombinant gene derived from Elizabethkingia meningoseptica.

PNGase F is a glycoamidase hydrolyzing the amide bond between the polypeptide asparagine and the innermost GlcNAc of all mammalian asparagine-linked complex, hybrid, or high mannose oligosaccharides. During the reaction, the asparagine residue from which the glycan is removed is deaminated to aspartic acid. The released oligosaccharide is left intact and can be used for further analysis. Removal of N-glycans is widely used for sample preparation for MS analysis – to reduce the protein heterogeneity and enable released glycan analysis – and to study the functional role of the N-glycan.

The glycoprotein sample is incubated with the Immobilized PNGase F resin in a spin column for 1 h to overnight using native reaction conditions or 15-60 min using denaturing reaction conditions. The deglycosylated glycoprotein is then easily collected by a centrifugation step. The activity of PNGase F on some glycoproteins can be slow or inhibited due to steric hindrance. Longer incubation times or denaturation of the glycoprotein may in these cases be required.

Unit Definition

0.05 ml of settled agarose resin hydrolyzes ≥ 95% of N-glycans from 0.2 mg etanercept in TBS, pH 8.6 in 1 h at 37°C as monitored by SDS-PAGE.

Content and Storage

Immobilized PNGase F Microspin columns contain sufficient material to remove N-glycans from 0.2 mg glycoprotein. The resin is supplied in 20% EtOH with no preservatives added, and are provided in packs of 5 or 10 columns. The columns are shipped cold, and should be stored at +4-8°C upon arrival. Do not freeze the product!

Immobilized PNGase F Denaturing contains microspin columns of Immobilized PNGase F (see above) and vials of RapiGest™* SF Surfactant. One 1 mg RapiGest™ SF vial is included per column of Immobilized PNGase F. RapiGest™ SF should be stored at room temperature. Once reconstituted in high purity water or a buffer (pH 7–10), the solution is stable for one week when stored at +2–8 °C.

Immobilized PNGase F is for R&D use only.
 


 
* RapiGest™ SF Surfactant from Waters Corporation is included in Immobilized PNGase F Denaturing. RapiGest™ is a trademark of Waters Corporation.

  • N-linked glycans on glycoproteins
  • 15+ min under denaturing conditions
    1+ h under native conditions
  • Denaturing or native conditions, RapiGest™ SF is included
  • Hydrolyzes the glycosidic bond between N-glycans and asparagine
  • G1-PF6-010

    Immobilized PNGase F 5 x 0.2 mg

    G1-PF6-010495.00Add to cart
  • G1-PF6-020

    Immobilized PNGase F 10 x 0.2 mg

    G1-PF6-020845.00Add to cart
  • G2-PDK-010

    Immobilized PNGase F Denaturing 5 x 0.2 mg

    G2-PDK-010685.00Add to cart
  • G2-PDK-020

    Immobilized PNGase F Denaturing 10 x 0.2 mg

    G2-PDK-0201,225.00Add to cart

Applications for Immobilized PNGase F

Product Documents

FAQ

Popular FAQ

The recommended buffer for best performance of the column is TBS buffer at pH 8.6. Other buffers with neutral pH containing 0.15 M NaCl can also be used but optimization may then be required.

 

Find all FAQ here

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Immobilized PNGase F Brochure

Immobilized PNGase F Brochure

Removal of N-glycans from glycoproteins.

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