Frequently Asked Questions
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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.
Learn more about GalactEXO®
How long is the reaction time for GalactEXO?
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.
Learn more about GalactEXO®
When and why should I use the OpeRATOR elution procedure?
The OpeRATOR enzyme digests O-glycosylated proteins N-terminally to the serine (S) and threonine (T) glycosylation sites. OpeRATOR therefore facilitates mapping of O-glycosylation sites by LC-MS and as the digested products no longer bind to the GlycOCATCH resin, elution and sample preparation can be achieved in a single step. However, this requires the O-glycosylation sites to be spaced closely together for the OpeRATOR digestion to yield peptides of a size suitable for analysis by LC-MS/MS.
Learn more about GlycOCATCH™
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 capacity of GlycOCATCH is lost.
Learn more about GlycOCATCH™
Can I load a digested protein solution directly onto the GlycOCATCH column?
Yes. IMPORTANT! Before you load your sample make sure that the protease in solution is inactivated and that the pH, buffer, chaotrope and NaCl concentrations are within the recommended ranges according to the instructions.
Learn more about GlycOCATCH™
Can OglyZOR be used to deglycosylate peptides generated with OpeRATOR?
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. The FucosEXO enzyme can be used for hydrolysis of α1-2, α1-3 and α1-4 linked fucose.
Learn more about GalactEXO®
Can I use FabRICATOR and PNGaseF in the same reaction?
Yes, PNGaseF and FabRICATOR can be used in a one-pot reaction as long as the reaction conditions follow the specifications for the FabRICATOR enzyme. It is important to have native reaction conditions for FabRICATOR to remain active.
Learn more about FabRICATOR®
Can I use FabRICATOR Fab2 Kit for generation and purification of fragments from Rabbit IgG?
FabRICATOR Fab2 Kit digests rabbit IgG but to capture the Fc fragments in the affinity step you need another resin than the CaptureSelect™ Fc normally included in the kit. Please contact the Genovis team if you are interested in processing rabbit IgG and we will provide columns with resin compatible with rabbit Fc in the FabRICATOR Fab2 Kit.
Learn more about FabRICATOR®
Can FabALACTICA and FabRICATOR be used in the same reaction?
Yes, for human IgG1 molecules both enzymes can be used in combination to generate a specific hinge peptide. The incubation time is significantly longer for FabALACTICA and it is recommended to first run the FabALACTICA digest and then add FabRICATOR for the last 30 min.
Learn more about FabALACTICA™, FabRICATOR®