Question 1

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 2

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 3

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 4

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 5

What is the smallest amount of IgG that I can process using the 250 ug kit?

Felicia Mejare · Accepted Answer

The GlyCLICK 250 ug kit is optimized for 250 ug of IgG as a starting material. We do not recommend using less than 100 ug of starting material in a minimum of 100µL to achieve proper end-over-end mixing on the GlycINATOR columns. Please contact support@genovis.com for assistance if you want to process smaller amounts then 100µg.

Question 6

What is the smallest amount of IgG that I can process using the 250 ug kit?

Felicia Mejare · Accepted Answer

The GlyCLICK 250 ug kit is optimized for 250 ug of IgG as a starting material. We do not recommend using less than 100 ug of starting material in a minimum of 100µL to achieve proper end-over-end mixing on the GlycINATOR columns. Please contact support@genovis.com for assistance if you want to process smaller amounts then 100µg.

Question 7

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 8

What is the smallest amount of IgG that I can process using the 250 ug kit?

Felicia Mejare · Accepted Answer

The GlyCLICK 250 ug kit is optimized for 250 ug of IgG as a starting material. We do not recommend using less than 100 ug of starting material in a minimum of 100µL to achieve proper end-over-end mixing on the GlycINATOR columns. Please contact support@genovis.com for assistance if you want to process smaller amounts then 100µg.

Question 9

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 10

How do I perform quality control of the conjugation process?

Felicia Mejare · Accepted Answer

Characterization using LC-MS will give a complete and clear picture of mass shift associated with successful conjugation if the antibody is analyzed reduced or fragmented. LC or LC-MS analysis of HC from a reduced sample or scFc fragments generated by FabRICATOR digestion provides distinct elution peaks for conjugated scFc or mass shifts corresponding to the degree of labelling (DOL) following conjugation. On the application page you can see an example of such analysis. Analysis of fluorescently labeled antibodies can also be performed by absorbance measurements and calculate the DOL using the Extinction coefficient for the antibody and the label and the correction factor for the label.

Question 11

What is the conjugation efficacy using GlyCLICK?

Felicia Mejare · Accepted Answer

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.

Question 12

What is the conjugation efficacy using GlyCLICK?

Felicia Mejare · Accepted Answer

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.

Question 13

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 14

Does TransGLYCIT work on any glycoprotein?

Jonathan Sjögren · Accepted Answer

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

Question 15

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 16

What antibody species can I transglycosylate using TransGLYCIT?

Jonathan Sjögren · Accepted Answer

The TransGLYCIT platform is developed for transglycosylation of human IgG.

Question 17

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 18

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 19

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 20

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 21

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 22

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.

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Scientific Posters

Antibody LC-MS Analysis

Fluorescent imaging

Applications

GlyCLICK

GlyCLICK Azide Activation

TransGLYCIT

Hydrolysis of Flexible Linkers

Sugar-free Proteins in a Few Hours

Antibody Oxidation