Is it possible to purchase Immobilized PNGaseF in bulk?

Yes! Contact us at info@genovis.com

Does Immobilized PNGase F remove all N-linked glycans?

Immobilized PNGAse F hydrolyzes the amide bond between the polypeptide asparagine and the innermost GlcNAc of all mammalian asparagine-linked complex, hybrid, or high mannose oligosaccharides. It does not remove N-glycans with α1-3 core fucosylation.

Is it possible the reuse the Immobilized PNGAse F column?

No, it is not recommended. We can only guarantee optimal performance for one-time use.

Can the denaturing protocol be performed at a lower temperature than 50 °C?

Yes, it is possible to perform the incubation at 37 °C if the incubation time is prolonged. Treat the protein sample mix containing 0.5% RapiGest at 90 °C, 5 min prior to the incubation with Immobilized PNGase F.

Can the deglycosylation on the column be performed under denaturing and reducing conditions?

The columns can be used for deglycosylation at native or denaturing conditions. Reducing conditions can be used with the denaturing protocol but the reaction parameters need to be optimized for the specific substrate.

Which buffers can be used with the Immobilized PNGaseF column?

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.

Can I use PBS 1X instead of the IgGZERO reaction buffer?

Yes, IgGZERO/deGlycIT are compatible with most commonly used buffers with pH ranging from 6.0 to 8.0, but the reaction conditions might need to be optimized to ensure optimal deglycosylation.

What is the difference between Immobilized GlycINATOR and deGlycIT?

In both products the enzyme is immobilized on agarose beads for IgG-specific hydrolysis of the Fc glycans to the innermost GlcNAc. The deGlycIT contains immobilized IgGZERO (EndoS) and Immobilized GlycINATOR contains GlycINATOR (EndoS2). The GlycINATOR enzyme has much higher enzymatic activity for high mannose and some bisected and hybrid glycans that can occur on mAbs.

Can I re-use the Immobilized GlycINATOR column?

We can only guarantee optimal deglycosylation for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.

Can I achieve Fc-specific deglycosylation on IgG with N-linked glycans in the Fab region?

Yes, the GlycINATOR enzyme binds specifically to the Fc region and therefore will not deglycosylate N-linked glycans in the Fab region.

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