Frequently Asked Questions

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Does the enzyme require the sample to be in the native fold or would denaturation help?

If digestion is insufficient, it could be caused by the O-glycans having a sterically inaccessible location on the sample that the enzyme cannot reach. In such cases, we recommend trying the following workflow: reduction, denaturation, carboxymethylation, rebuffering, and then digestion with SialEXO and OpeRATOR.

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Can OpeRATOR digest consecutive O-glycosylated sites fully?

The relative abundance and site occupancy of O-glycosylation may vary a lot in a glycoprotein sample, and this will result in the generation of peptides with variable sizes. In some cases, the peptides contain more than one O-glycan, i.e. OpeRATOR does not digest at every site in every molecule. However, the missed digestion sites differ from molecule to molecule, and the compiled data will provide valuable information on the O-glycosylated sites.

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Will OpeRATOR work on simple hexose or pentose?

No, the enzyme recognizes mucin type glycosylation with an inner GalNAc linked to a hexose sugar.

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Are there any known inhibitors of OpeRATOR?

OpeRATOR is a metalloprotease and as such highly sensitive to chelating agents such as EDTA. Concentrations >5mM lead to complete inhibition of the enzyme. OpeRATOR activity is also moderately inhibited by ZnCl₂.

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Can I use OglyZOR and PNGaseF in the same reaction?

Yes, we have performed successful experiments with OglyZOR, SialEXO and PNGaseF in the same reaction under native conditions O/N at pH ~7.0. If denaturing reaction conditions are required for PNGaseF, the reaction with OglyZOR and SialEXO needs to be performed first under native conditions, then add PNGaseF and the required buffer.

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Will OglyZOR leave the N-glycans intact?

OglyZOR only works on desialylated O-glycans therefore is a sialidase (SialEXO) included with the product. SialEXO is not O-glycan specific and will therefore desialylate any N-glycans that are present.

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Will OglyZOR work on extended core 1 structures or single HexNAc?

No, OglyZOR recognizes the core 1 disaccharide HexNAcHex (or GalNAcGal) and to some extend also the core 3 disaccharide HexNAc(2) (or GalNAcGlcNAc).

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I read that cleavage buffer for IgGZERO is 10 mM sodium phosphate, 150 mM NaCl, pH 7.4 Can I use PBS 1X in place of cleavage buffer?

Yes, many buffers are compatible with IgGZERO. Please keep pH close to neutral.

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I did not get complete deglycosylation using IgGZERO Immobilized, how can I improve digestion?

First you could increase incubation time and increase temperature to 37°C. One explanation for incomplete deglycosylation with IgGZERO Immobilized could be that the antibody has higher amounts of high mannose, hybrid glycans or bisected glycans. For these glycans IgGZERO has low or no activity. High mannose glycans are particularly resistant to digestion recommend you try our other endoglycosidase GlycINATOR (EndoS2). This enzyme has much higher activity on these types of glycans sometimes seen in mAbs.

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Is cysteine really needed for activation of the cysteine protease if TCEP or DTT is added in the reaction?

Yes, cysteine is required for both activity and specificity of the GingisREX enzyme.

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