Question 1

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 2

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 3

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 4

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.

Question 5

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.

Question 6

Can I use FabRICATOR-HPLC in a 2D-LC setting?

kk · Accepted Answer

Yes, it is possible to digest the antibody in the first dimension and then separate the subunits in the second dimension. Please see the application note for detailed instructions.

Question 7

Which digestion buffers are compatible with FabRICATOR-HPLC?

kk · Accepted Answer

We have tested PBS and 150 mM ammonium acetate, pH7 with good results. More buffers were found to be compatible with FabRICATOR digestion in solution (more info) and should be suitable for FabRICATOR-HPLC as well. However, the ionic strength of the buffer influences retention of the antibody fragments on the FabRICATOR-HPLC column and a minimum of 100-150 mM salt is necessary for IgG1 subunits to elute as a single, narrow peak. Other IgG subclasses or fusion proteins might need some optimization of buffer composition.

Question 8

Can I process diluted IgG samples using FabRICATOR-HPLC?

kk · Accepted Answer

We have tested concentrations as low 0.025 mg/ml without any measurable effect on digestion efficiency. However, low concentration samples require a longer injection time and consequently lead to a broader elution peak from the FabRICATOR-HPLC column.

Question 9

What is the FabRICATOR-HPLC?

kk · Accepted Answer

FabRICATOR-HPLC is an HPLC column with the FabRICATOR (IdeS) protease immobilized on a HPLC compatible matrix for automated antibody subunit generation.

Question 10

How many samples can I run through a column?

kk · Accepted Answer

The FabRICATOR-HPLC can run >400 samples of 10 ug of IgG.

Question 11

How many samples can I run through a column?

kk · Accepted Answer

The FabRICATOR-HPLC can run >400 samples of 10 ug of IgG.

Question 12

There are not yet any FAQ's for this product

kk · Accepted Answer

If you have a technical question, please submit a support case.

Question 13

There are not yet any FAQ's for this product

kk · Accepted Answer

If you have a technical question, please submit a support case.

Question 14

Can I use FabRICATOR MagIC in a automation system?

kk · Accepted Answer

FabRICATOR MagIC is suitable for using in automated systems that can handle magnetic beads. On the applications page you can read about how it’s used in a KingFisher™ workflow.

Question 15

What is the FabRICATOR MagIC?

kk · Accepted Answer

The FabRICATOR MagIC magnetic beads with immobilized FabRICATOR (IdeS) enzyme.

Question 16

Which digestion buffers are compatible with FabRICATOR MagIC?

kk · Accepted Answer

The protocol for FabRICATOR MagIC is based on using standard PBS pH 7.4 as digestion buffer. FabRICATOR in solution is compatible with most commonly used buffers at pH=6-8 and the same buffers should work for FabRICATOR MagIC, but the incubation time might need to be optimized.

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

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 19

Can I use FabRICATOR MagIC in a automation system?

kk · Accepted Answer

FabRICATOR MagIC is suitable for using in automated systems that can handle magnetic beads. On the applications page you can read about how it’s used in a KingFisher™ workflow.

Question 20

Can I use FabRICATOR-HPLC in a 2D-LC setting?

kk · Accepted Answer

Yes, it is possible to digest the antibody in the first dimension and then separate the subunits in the second dimension. Please see the application note for detailed instructions.

Question 21

What is the FabRICATOR MagIC?

kk · Accepted Answer

The FabRICATOR MagIC magnetic beads with immobilized FabRICATOR (IdeS) enzyme.

Question 22

Which digestion buffers are compatible with FabRICATOR MagIC?

kk · Accepted Answer

The protocol for FabRICATOR MagIC is based on using standard PBS pH 7.4 as digestion buffer. FabRICATOR in solution is compatible with most commonly used buffers at pH=6-8 and the same buffers should work for FabRICATOR MagIC, but the incubation time might need to be optimized.

Question 23

Which digestion buffers are compatible with FabRICATOR-HPLC?

kk · Accepted Answer

We have tested PBS and 150 mM ammonium acetate, pH7 with good results. More buffers were found to be compatible with FabRICATOR digestion in solution (more info) and should be suitable for FabRICATOR-HPLC as well. However, the ionic strength of the buffer influences retention of the antibody fragments on the FabRICATOR-HPLC column and a minimum of 100-150 mM salt is necessary for IgG1 subunits to elute as a single, narrow peak. Other IgG subclasses or fusion proteins might need some optimization of buffer composition.

Question 24

Which digestion buffers are compatible with FabRICATOR-HPLC?

kk · Accepted Answer

We have tested PBS and 150 mM ammonium acetate, pH7 with good results. More buffers were found to be compatible with FabRICATOR digestion in solution (more info) and should be suitable for FabRICATOR-HPLC as well. However, the ionic strength of the buffer influences retention of the antibody fragments on the FabRICATOR-HPLC column and a minimum of 100-150 mM salt is necessary for IgG1 subunits to elute as a single, narrow peak. Other IgG subclasses or fusion proteins might need some optimization of buffer composition.

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