Does ImpaRATOR cleave at all O-glycosylated sites?
ImpaRATOR has a broad activity towards different O-glycan structures; however, the enzyme has limited activity towards sites with two adjacent O-glycosylated Ser/Thr residues. For complete information about O-glycan sites in glycoprotein substrates containing several sites with two adjacent O-glycosylated Ser/Thr residues, OpeRATOR may be a better option.
Can ImpaRATOR be used under denaturing conditions?
Insufficient digestion during non-denaturing conditions can be caused by O-glycosylation sites within the glycoprotein inaccessible to the enzyme. In such cases, we recommend trying the following workflow: reduction, denaturation, carboxymethylation, buffer-exchange, and then digestion with ImpaRATOR. If detergent is added, make sure to include a detergent removal step prior ImpaRATOR digestion.
Are there any known inhibitors of ImpaRATOR?
ImpaRATOR is a metalloprotease and thereby sensitive to chelating agents such as EDTA. Concentrations > 1 mM EDTA results in complete inhibition of the enzyme. In addition, the ImpaRATOR activity is inhibited by reducing agents and detergents.
Is it possible to reuse the OmniGLYZOR column?
No, it is not recommended. We can only guarantee optimal performance for one-time use.
Does OmniGLYZOR remove all N-glycans?
OmniGLYZOR 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.
Can OmniGLYZOR be used for released glycan analysis?
No, both N- and O-glycans are trimmed by the exoglycosidases present in OmniGLYZOR and do not longer represent the structures found on the intact glycoprotein substrate.
Does PNGase F Immobilized remove all N-linked glycans?
PNGase F Immobilized 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 PNGAse F Immobilized column?
No, it is not recommended. We can only guarantee optimal performance for one-time use.
Can the deglycosylation on the column be performed under denaturing conditions?
The columns are recommended to be used for deglycosylation at native conditions only.