Does GalactEXO digest fucosylated glycan structures such as Lewis X or Lewis a?

No. Branching fucose linked to the same residue as the β-galactose inhibits GalactEXO activity and needs to be removed prior to GalactEXO treatment.

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Can OglyZOR be used to deglycosylate peptides generated with OpeRATOR?

Yes, this workflow enables MS/MS of O-glycosylated peptides with removed O-glycans.

Can I load a digested protein solution directly onto the GlycOCATCH column?

Before you load your sample, make sure that the protease in the solution is inactivated, and that the pH, buffer, chaotrope and NaCl concentrations are within the recommended ranges (according to the instructions).

Is it possible to digest the glycoprotein with OpeRATOR first and then enrich the O-glycopeptides on GlycOCATCH?

No, when the glycoprotein is digested with OpeRATOR, the binding ability of GlycOCATCH is lost.

When and why should I use the OpeRATOR elution procedure?

The OpeRATOR enzyme digests O-glycosylated proteins N-terminally of the serine (S) and threonine (T) glycosylation sites. If you want to know at which S or T a specific O-glycan is sited, we recommend elution with OpeRATOR. We also recommend elution with OpeRATOR if you have a glycoprotein with suitable distances between the O-glycan sites and suitable distances between the O-glycans and the N/C-terminals, and you want to generate peptides of suitable size for your purpose.

How long is the reaction time?

For most substrates such as antibodies or Fc-fusion proteins a 2 h incubation results in hydrolysis of over 95% of the present galactose. More complex substrates with high degree of galactosylation may require longer incubation times.

Does GalactEXO hydrolyze galactose on N and O-glycosylated proteins?

Yes, both beta1-3 linked galactose on O-glycans and beta1-4 galactose on N-glycans are hydrolyzed by GalactEXO.

Can you combine GalactEXO with SialEXO or SialEXO 23 to remove both galactoses and sialic acids in a single reaction?

Yes, you can. All three enzymes perform well in the same reaction buffer (20 mM Tris pH 6.8). There is no need to increase incubation time when combining the enzymes.

We want to make Fab’s directly from antibodies. When would the selection of FabALACTICA be the first choice over GingisKHAN or FabULOUS?

FabALACTICA does not require reducing conditions as both GingisKHAN and FabULOUS does, and allows for generation of Fab fragments from native human IgG1.