Q: There are not yet any FAQ's for this product

If you have a technical question, please contact us .

Q: There are not yet any FAQ's for this product

If you have a technical question, please contact us .

Question 1

There are not yet any FAQ's for this product

kk · Accepted Answer

If you have a technical question, please contact us.

Question 2

There are not yet any FAQ's for this product

kk · Accepted Answer

If you have a technical question, please contact us.

Question 3

Is it possible to use FabRICATOR in a buffer containing EDTA or HSA?

kk · Accepted Answer

Yes. FabRICATOR is compatible with EDTA. Make sure your pH is within 6-8.

Question 4

Does FabRICATOR (IdeS) have protease activity on IgM?

kk · Accepted Answer

No. According to the following reference, FabRICATOR does not digest IgM. Pavel-Rammingen et al., 2002. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G.

Question 5

Is FabRICATOR stable in the presence of denaturing agents such as guanidine hydrochloride or SDS?

kk · Accepted Answer

We have tested 4-6 M GuHCl which the enzyme does not withstand without significant decrease of activity. SDS have not been evaluated but 0.1% RapiGest from waters have negative influence on activity. Taken together the enzyme does not well tolerate denaturing agents although we have not tested this in more detailed studies.

Question 6

I have a small modification in the sequence below the hinge, will FabRICATOR still digest my IgG?

kk · Accepted Answer

FabRICATOR is a very specific enzyme and small changes in the sequence in or around the digestion site (...CPAPELLG / GPSVF...) may reduce or completely impair the ability to digest the IgG. It is very difficult to predict the outcome for each specific modification. It might work, but we cannot guarantee it unfortunately, the only way of knowing is to perform a digestion test. Some suggestions that are worth testing if you experience incomplete digestion: Increase the incubation time, increase the amount of added enzyme, use a PBS with greatly reduced salt concentration (5-10 mM instead of 140 mM) and pH 6.5 instead of 7.4.

Question 7

Can I cleave Fc-fusion proteins with FabRICATOR?

kk · Accepted Answer

Yes, FabRICATOR should be able to digest a Fc fusion protein as long as you have not fused the protein too close to the cleavage site. FabRICATOR digests human IgG just below the hinge (...CPAPELLG / GPSVF...), leaving the hinge on the fusion-part.

Question 8

Can I cleave Fc-fusion proteins with FabRICATOR?

kk · Accepted Answer

Yes, FabRICATOR should be able to digest a Fc fusion protein as long as you have not fused the protein too close to the cleavage site. FabRICATOR digests human IgG just below the hinge (...CPAPELLG / GPSVF...), leaving the hinge on the fusion-part.

Question 9

Can I re-use FabALACTICA Immobilized columns?

Felicia Mejare · Accepted Answer

We can only guarantee optimal digestion for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.

Question 10

Can I digest Fc-fusion proteins using FabALACTICA (IgdE)?

Felicia Mejare · Accepted Answer

No, FabALACTICA has shown very poor activity on Fc-fusion proteins.

Question 11

Can I release Fc fragments from the CaptureSelect column after isolating the Fab fragments?

Felicia Mejare · Accepted Answer

It is not recommended to elute the Fc fragments from the CaptureSelect column since the fraction might also include undigested or semi digested material.

Question 12

Can FabALACTICA and FabRICATOR be used in the same reaction?

Felicia Mejare · Accepted Answer

Yes, for human IgG1 molecules both enzymes can be used in combination to generate a specific hinge peptide. The incubation time is significantly longer for FabALACTICA and it is recommended to first run the FabALACTICA digest and then add FabRICATOR for the last 30 min.

Question 13

How can I remove residual enzyme from the digested antibody in solution?

Felicia Mejare · Accepted Answer

We recommend using FabALACTICA Immobilized for digesting human IgG1 and generating a final preparation free of any residual enzyme. You can also use FabALACTICA Fab kit for digestion of human IgG1 and purification of the generated Fab fragments.

Question 14

How can I remove residual enzyme from the digested antibody in solution?

Felicia Mejare · Accepted Answer

We recommend using FabALACTICA Immobilized for digesting human IgG1 and generating a final preparation free of any residual enzyme. You can also use FabALACTICA Fab kit for digestion of human IgG1 and purification of the generated Fab fragments.

Question 15

Can I use FabRICATOR Fab2 Kit for generation and purification of fragments from Rabbit IgG?

Felicia Mejare · Accepted Answer

FabRICATOR Fab2 Kit digests rabbit IgG but to capture the Fc fragments in the affinity step you need another resin than the CaptureSelect&trade; Fc normally included in the kit. Please contact the Genovis team if you are interested in processing rabbit IgG and we will provide columns with resin compatible with rabbit Fc in the FabRICATOR Fab2 Kit.

Question 16

Can I release Fc fragments from the CaptureSelect column after isolating the F(ab')2 fragments?

kk · Accepted Answer

It is possible to elute the Fc fragments from the CaptureSelect column using a low pH buffer, for example a 0.1M Glycine buffer pH=3.0. The pH must immediately after elution be adjusted to neutral pH by adding 0.1x elution volume of 1M Tris pH=8.0.

Question 17

Does FabRICATOR introduce modifications?

kk · Accepted Answer

FabRICATOR is a cysteine protease. It should not result in any other modifications than the digestion of the peptide bond. Digestion will be between the two Glycines (...CPAPELLG / GPSVF...) and the C-terminus will regain the carboxyl group - that is addition of one OH-group and the N-terminal amino group will be regained (addition of one H).

In summary, R1-CO-NH-R2 + H2O --> R1-COOH + NH2-R2.

Question 18

Can I use protein A or MabSelectSure (GE) for removal of Fc-fragments?

kk · Accepted Answer

Genovis has chosen CaptureSelect in the FabRICATOR Fab2 Kit for removal of Fc fragments. CaptureSelect has a very high specific affinity for the Fc. Both protein A and MabSelect Sure (GE) may have affinity for the Fab which may result in a lower recovery of the generated F(ab’)2 fragments and the degree of affinity is antibody dependent. We have many customer reports on this subject, and they usually come back to us for the CaptureSelect columns for the above reason.

Question 19

Can I re-use FabRICATOR Immobilized columns?

kk · Accepted Answer

We can only guarantee optimal digestion for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.

Question 20

Can I re-use FabRICATOR Immobilized columns?

kk · Accepted Answer

We can only guarantee optimal digestion for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.

Question 21

Can I use PBS 1X instead of the IgGZERO reaction buffer?

Felicia Mejare · Accepted Answer

Yes, the IgGZERO enzyme is compatible with most commonly used buffers with pH ranging from 6.0 to 8.0, but the reaction conditions might need to be optimized to ensure optimal deglycosylation.

Question 22

I read that cleavage buffer for IgGZERO is 10 mM sodium phosphate, 150 mM NaCl, pH 7.4 Can I use PBS 1X in place of cleavage buffer?

kk · Accepted Answer

Yes, many buffers are compatible with IgGZERO. Please keep pH close to neutral.

Question 23

I did not get complete deglycosylation using IgGZERO Immobilized, how can I improve digestion?

kk · Accepted Answer

First you could increase incubation time and increase temperature to 37°C. One explanation for incomplete deglycosylation with IgGZERO Immobilized could be that the antibody has higher amounts of high mannose, hybrid glycans or bisected glycans. For these glycans IgGZERO has low or no activity. High mannose glycans are particularly resistant to digestion recommend you try our other endoglycosidase GlycINATOR (EndoS2). This enzyme has much higher activity on these types of glycans sometimes seen in mAbs.

Question 24

What is the difference between GlycINATOR Immobilized and IgGZERO Immobilized?

Felicia Mejare · Accepted Answer

In both products the enzyme is immobilized on agarose beads for IgG-specific hydrolysis of the Fc glycans to the innermost GlcNAc. IgGZERO Immobilized contains IgGZERO (EndoS) and GlycINATOR Immobilized contains GlycINATOR (EndoS2). The GlycINATOR enzyme has much higher enzymatic activity for high mannose and some bisected and hybrid glycans that can occur on mAbs.

Question 25

What is the difference between GlycINATOR and IgGZERO?

Felicia Mejare · Accepted Answer

IgGZERO (EndoS) and GlycINATOR (EndoS2) are both IgG-specific endoglycosidases that hydrolyses Fc glycans to the innermost GlcNAc. The GlycINATOR enzyme has a broader substrate selectivity and therefore much higher enzymatic activity on high mannose and some bisected and hybrid glycans that can occur on mAbs. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

Question 26

What is the difference between GlycINATOR and IgGZERO?

Felicia Mejare · Accepted Answer

IgGZERO (EndoS) and GlycINATOR (EndoS2) are both IgG-specific endoglycosidases that hydrolyses Fc glycans to the innermost GlcNAc. The GlycINATOR enzyme has a broader substrate selectivity and therefore much higher enzymatic activity on high mannose and some bisected and hybrid glycans that can occur on mAbs. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.

Question 27

There are not yet any FAQ's for this product

kk · Accepted Answer

If you have a technical question, please contact us.

Question 28

There are not yet any FAQ's for this product

kk · Accepted Answer

If you have a technical question, please contact us.

Question 29

Is it possible to reuse the OmniGLYZOR column?

Maria Ekemohn · Accepted Answer

No, it is not recommended. We can only guarantee optimal performance for one-time use.

Question 30

Can OmniGLYZOR be used for released glycan analysis?

Maria Ekemohn · Accepted Answer

No, both N- and O-glycans are trimmed by the exoglycosidases present in OmniGLYZOR and do not longer represent the structures found on the intact glycoprotein substrate.

Question 31

Does OmniGLYZOR remove all N-glycans?

Maria Ekemohn · Accepted Answer

OmniGLYZOR hydrolyzes the amide bond between the polypeptide asparagine and the innermost GlcNAc of all mammalian asparagine-linked complex, hybrid, or high mannose oligosaccharides. It does not remove N-glycans with α1-3 core fucosylation.

Question 32

Does OmniGLYZOR remove all N-glycans?

Maria Ekemohn · Accepted Answer

OmniGLYZOR hydrolyzes the amide bond between the polypeptide asparagine and the innermost GlcNAc of all mammalian asparagine-linked complex, hybrid, or high mannose oligosaccharides. It does not remove N-glycans with α1-3 core fucosylation.

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Resources

FabDELLO

Hydrolysis of Flexible Linkers

FabRICATOR Lyophilized

FabALACTICA

FabRICATOR Fab2 Kit

IgGZERO

Anti-FabRICATOR

OmniGLYZOR

Antibody LC-MS Analysis

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