Frequently Asked Questions

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.

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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.

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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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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.

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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.

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

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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.

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No, GlycOCATCH is designed to bind specifically to mucin type (core 1) O-glycosylated proteins and peptides. The performance is significantly enhanced by removal of sialic acids using SialEXO.

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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.

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

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