Powered by BiozSite-specific Conjugation of IgG
GlyCLICK is a three-step conjugation technology for site-specific and quantitative conjugation of IgG from several species and subclasses.
Specific Fc N-glycan hydrolysis allows for site-specific conjugation at the core GlcNAcs using robust click-chemistry and results in a degree of label (DOL) or drug-antibody ratio (DAR) of 2. The reliable performance ensures quantitative conjugation and intact immunoreactivity for sensitive applications.
The technology is available in a range of kit formats, including conjugation with fluorophores, biotin, DFO, or toxins for ADC development. The format "Azide Activation" renders the antibody azide-activated, for conjugation with any alkyne-reactive label of choice.
- Site-specific conjugation of IgG
- Reliable performance ensuring quantitative conjugation and preserved immunoreactivity for sensitive applications
- Stable and homogenous conjugation generates a degree of labeling (DOL) of 2
GlyCLICK® Fluorophore
Site-specific conjugation of IgG with Alexa Fluor® 488, 555 or 647GlyCLICK® Biotin
Site-specific conjugation of IgG with biotinGlyCLICK® DFO
Site-specific conjugation of IgG with DFOGlyCLICK® ADC
Site-specific conjugation of IgG with MMAE or PNUGlyCLICK® Azide Activation
Site-specific conjugation of IgG with azide-alkyne click chemistryAbout GlyCLICK®
How GlyCLICK works
- Deglycosylation
The Fc specific endoglycosidase GlycINATOR® (EndoS2) hydrolyzes the Fc glycans to the inner most GlcNAc moiety on several subclasses and species of IgG. The enzyme removes all glycoforms, including; high-mannose, hybrid, complex, and bisecting type glycans. - Azide Activation
Azide-containing UDP-GalNAz is enzymatically attached to the exposed GlcNAc using the ß-1,4-Galactosyltransferase Y289L* (GalT) to generate an azide-activated antibody that is reactive with an alkyne-carrying label. - CLICK Reaction
The azide activated antibody is conjugated with a cyclooctyne-functionalized label of choice in a biorthogonal reaction through strain-promoted copper-free click chemistry (SPAAC) to form a stable triazole.
Applications
Introduction to Conjugation
Fluorescence Microscopy
ADC Development
PET/SPECT Imaging
Flow CytometryGlyCLICK® Citations
FAQ and Support
Popular FAQ
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.
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.
Problems can arise from freezing of the conjugates. We cannot guarantee the quality after freezing and therefore recommend storage at +4°C.
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.
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.
Support