Frequently Asked Questions
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Can I digest Fc-fusion proteins using FabULOUS (SpeB)?
Yes, FabULOUS should be able to digest a Fc fusion protein as long as you have not fused the protein too close to the cleavage site. FabULOUS digests human IgG1 just above the hinge (…KTHT / CPPCP…).
Learn more about FabULOUS™
Can I purify Fab fragments from human IgG with FabULOUS Fab kit?
No, FabULOUS Fab kit contains CaptureSelect LC-kappa (mur) spin columns with selective affinity for murine LC-kappa. If you need to generate pure Fab from human IgG1 you can use either FabALACTICA Fab Kit or GingisKHAN Fab Kit. If you have a different subclass of human IgG, please contact support@genovis.com for guidance.
Learn more about FabULOUS™
I work with mouse IgG2a containing LC-lambda, can I use another affinity resin for purification of the Fab?
Yes. There is the possibility to exchange the CaptureSelect LC-kappa (mur) columns to columns containing CaptureSelect LC-lambda (mouse). This resin is specific for mouse IgG. Please contact support@genovis.com if this is of interest.
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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.
Learn more about GlycINATOR™, IgGZERO™
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.
Learn more about GlycINATOR™, IgGZERO™
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.
Learn more about GlycINATOR™
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.
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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.
Learn more about GlycINATOR™
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.
Learn more about IgGZERO™
What is the conjugation efficacy using GlyCLICK?
All the steps (deglycosylation, azide activation and click reaction) in GlyCLICK are highly efficient. Normally, all molecules are conjugated when using GlyCLICK, resulting in DOL=2. On the application page we have shown by MS-analysis that all molecules have been conjugated and there is no remaining un-conjugated material.
Learn more about GlyCLICK®