GlyCLICK Fluorophore

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.

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.

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.

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.