Does TransGLYCIT work on any glycoprotein?

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

What is the substrate specificity of GlycINATOR?

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

What antibody species can I transglycosylate using TransGLYCIT?

The TransGLYCIT platform is developed for transglycosylation of human IgG.

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

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

Does TransGLYCIT work on all human IgG subclasses?

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.

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

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.

Can I freeze antibodies after biotinylation using GlyCLICK?

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

What fluorescent labels can I use with the GlyCLICK technology?

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.

How do I perform quality control of the conjugation process?

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.

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

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.