Question 1

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 2

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 3

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 4

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 5

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 6

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 7

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 8

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 9

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 10

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 11

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 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

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 14

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 15

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 16

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 17

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 18

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 19

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 20

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 21

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 22

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 23

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 24

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 25

Does TransGLYCIT work on any glycoprotein?

Jonathan Sjögren · Accepted Answer

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

Question 26

What antibody species can I transglycosylate using TransGLYCIT?

Jonathan Sjögren · Accepted Answer

The TransGLYCIT platform is developed for transglycosylation of human IgG.

Question 27

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 28

Does GalactEXO digest fucosylated glycan structures such as Lewis X or Lewis a?

kk · Accepted Answer

No. Branching fucose linked to the same residue as the β-galactose inhibits GalactEXO activity and needs to be removed prior to GalactEXO treatment. The FucosEXO enzyme can be used for hydrolysis of α1-2, α1-3 and α1-4 linked fucose.

Question 29

Does GalactEXO hydrolyze galactose on N and O-glycosylated proteins?

kk · Accepted Answer

Yes, both beta1-3 linked galactose on O-glycans and beta1-4 galactose on N-glycans are hydrolyzed by GalactEXO.

Question 30

How long is the reaction time for GalactEXO?

kk · Accepted Answer

For most substrates such as antibodies or Fc-fusion proteins a 2 h incubation results in hydrolysis of over 95% of the present galactose. More complex substrates with high degree of galactosylation may require longer incubation times.

Question 31

Can you combine GalactEXO with SialEXO or SialEXO 23 to remove both galactoses and sialic acids in a single reaction?

kk · Accepted Answer

Yes, you can. All three enzymes perform well in the same reaction buffer (20 mM Tris pH 6.8). There is no need to increase incubation time when combining the enzymes.

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Analysis of Afucosylation Levels

GlyCLICK Fluorophore

GlyCLICK Biotin

GlyCLICK DFO

Fluorescent imaging

GlyCLICK

GlyCLICK Azide Activation

TransGLYCIT

GalactEXO