Can I use PBS 1X instead of the IgGZERO reaction buffer?
Yes, the IgGZERO enzyme is compatible with most commonly used buffers with pH ranging from 6.0 to 8.0, but the reaction conditions might need to be optimized to ensure optimal deglycosylation.
What is the difference between GlycINATOR Immobilized and IgGZERO Immobilized?
In both products the enzyme is immobilized on agarose beads for IgG-specific hydrolysis of the Fc glycans to the innermost GlcNAc. IgGZERO Immobilized contains IgGZERO (EndoS) and GlycINATOR Immobilized contains GlycINATOR (EndoS2). The GlycINATOR enzyme has much higher enzymatic activity for high mannose and some bisected and hybrid glycans that can occur on mAbs.
Can I re-use the GlycINATOR Immobilized column?
We can only guarantee optimal deglycosylation for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.
Can I achieve Fc-specific deglycosylation on IgG with N-linked glycans in the Fab region?
Yes, the GlycINATOR enzyme binds specifically to the Fc region and therefore will not deglycosylate N-linked glycans in the Fab region.
What is the difference between GlycINATOR and IgGZERO?
IgGZERO (EndoS) and GlycINATOR (EndoS2) are both IgG-specific endoglycosidases that hydrolyses Fc glycans to the innermost GlcNAc. The GlycINATOR enzyme has a broader substrate selectivity and therefore much higher enzymatic activity on high mannose and some bisected and hybrid glycans that can occur on mAbs. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.
Which should I choose, GlycINATOR or IgGZERO?
Typically, GlycINATOR is the first choice since it displays enzymatic activity on all glycoforms present on IgG, including complex type, high mannose, bisected and hybrid type glycans. IgGZERO does not hydrolyze high mannose glycans. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.
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.
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.