Frequently Asked Questions
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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®
Can I digest all mouse IgG subclasses with FabRICATOR Z?
No, FabRICATOR Z only digests mouse IgG2a (….CPAPNLLG / GPSVF.. ) and IgG3 (…CPAPNILG / GPSVF…) at one specific site below the hinge. If Fab fragments are an option, we recommend our FabULOUS enzyme that digest mouse IgG1 above the hinge. FabULOUS requires light reducing conditions for optimal activity. If strong reducing conditions are used, this may likely cause reduction of the interchain disulfides.
Learn more about FabRICATOR® Z
Can I digest rat IgG2a with FabRICATOR Z?
Unfortunately, FabRICATOR Z does not digest rat IgG2a. If generating Fab fragments is an option, we suggest using FabULOUS enzyme that digests rat IgG2a. FabULOUS does not have a single specific cleavage site, but a primary digestion site, so there can be more than one digestion site. The digestion site(s) for rat IgG2a using FabULOUS is not determined, but the generated fragments are Fab and Fc.
Learn more about FabRICATOR® Z