Question 1

Is it possible to reuse the OmniGLYZOR column?

Maria Ekemohn · Accepted Answer

No, it is not recommended. We can only guarantee optimal performance for one-time use.

Question 2

Can OmniGLYZOR be used for released glycan analysis?

Maria Ekemohn · Accepted Answer

No, both N- and O-glycans are trimmed by the exoglycosidases present in OmniGLYZOR and do not longer represent the structures found on the intact glycoprotein substrate.

Question 3

Does OmniGLYZOR remove all N-glycans?

Maria Ekemohn · Accepted Answer

OmniGLYZOR hydrolyzes the amide bond between the polypeptide asparagine and the innermost GlcNAc of all mammalian asparagine-linked complex, hybrid, or high mannose oligosaccharides. It does not remove N-glycans with α1-3 core fucosylation.

Question 4

Does OmniGLYZOR remove all N-glycans?

Maria Ekemohn · Accepted Answer

OmniGLYZOR hydrolyzes the amide bond between the polypeptide asparagine and the innermost GlcNAc of all mammalian asparagine-linked complex, hybrid, or high mannose oligosaccharides. It does not remove N-glycans with α1-3 core fucosylation.

Question 5

Can I use the FucosEXO 16 enzyme on native antibodies to remove the core fucose?

Jonathan Sjögren · Accepted Answer

Unfortunately no, the enzyme requires trimming of the Fc-glycan using GlycINATOR to enable access to the core fucose substrate.

Question 6

Does TransGLYCIT work on all human IgG subclasses?

Jonathan Sjögren · Accepted Answer

Yes, the transglycosylation reaction can be performed on all subclasses of human IgG. The reaction is somewhat slower on IgG2 and longer incubation times may be necessary to obtain over 95% transglycosylation.

Question 7

Does TransGLYCIT work on any glycoprotein?

Jonathan Sjögren · Accepted Answer

No, TransGLYCIT is based on IgG specific enzymes and will only transglycosylate IgG.

Question 8

What is the substrate specificity of GlycINATOR?

Jonathan Sjögren · Accepted Answer

GlycINATOR is an IgG specific endoglycosidase that hydrolyzes complex, hybrid and high mannonse type glycans on the conserved Fc site on IgG.

Question 9

What antibody species can I transglycosylate using TransGLYCIT?

Jonathan Sjögren · Accepted Answer

The TransGLYCIT platform is developed for transglycosylation of human IgG.

Question 10

What antibody species can I transglycosylate using TransGLYCIT?

Jonathan Sjögren · Accepted Answer

The TransGLYCIT platform is developed for transglycosylation of human IgG.

Question 11

What fluorescent labels can I use with the GlyCLICK technology?

Felicia Mejare · Accepted Answer

GlyCLICK is available in kit formats with AlexaFluor®488, AlexaFluor®555, AlexaFluor®647 or in the Azide Activation kit format for conjugation of a label of choice. With the fluorescent label being alkyne-modified (carrying for example DIBO, DBCO or BCN) it is possible to combine it with the GlyCLICK azide activation kit.

Question 12

Which isotopes can I use to radiolabel IgG after chelation using GlyCLICK?

Felicia Mejare · Accepted Answer

The GlyCLICK technology can be used to conjugate IgG with Desferrioxamine (DFO) using the GlyCLICK DFO kit. The DFO is a chelating agent for radiolabeling with the radioisotope Zirconium-89 (89Zr) for PET-imaging. The Azide Activation kit can be used to conjugate a chelator of choice if it is alkyne-modified (carrying for example DIBO, DBCO or BCN) to be compatible with the click chemistry.

Question 13

Can I freeze antibodies after biotinylation using GlyCLICK?

Felicia Mejare · Accepted Answer

Problems can arise from freezing of the conjugates. We cannot guarantee the quality after freezing and therefore recommend storage at +4°C.

Question 14

For how long can I store azide activated antibodies?

Felicia Mejare · Accepted Answer

The stability of azide activated material varies depending on the antibody and if stabilizing agents have been added. In our labs we have stored azide activated antibodies in TBS buffer without any additives at +4°C for over two years. The azide activation was still OK, and the click reaction generated antibodies with DOL=2. If azide is added as a preservative during long-term storage, it must be removed prior to click reaction.

Question 15

Why do I need to use an alkyne-functionalized label with GlyCLICK?

Felicia Mejare · Accepted Answer

The final step of GlyCLICK labeling kit is based on a copper free click reaction (SPAAC) and it is a strain promoted reaction between an azide and an alkyne-carrying label. As the name indicates the reaction occurs spontaneously due to the strained bond angles. The use of this click reaction that forms stable conjugates, allows you to freely choose label or payload to combine with your IgG as long as it is alkyne-functionalized. Several alkyne-carrying labels are commercially available with cyclooctyne structures such as DIBO, DBCO or BCN.

Question 16

Why do I need to use an alkyne-functionalized label with GlyCLICK?

Felicia Mejare · Accepted Answer

The final step of GlyCLICK labeling kit is based on a copper free click reaction (SPAAC) and it is a strain promoted reaction between an azide and an alkyne-carrying label. As the name indicates the reaction occurs spontaneously due to the strained bond angles. The use of this click reaction that forms stable conjugates, allows you to freely choose label or payload to combine with your IgG as long as it is alkyne-functionalized. Several alkyne-carrying labels are commercially available with cyclooctyne structures such as DIBO, DBCO or BCN.

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