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

Is FabULOUS working on human IgG4?

kk · Accepted Answer

Yes, but a longer incubation time is needed.

Question 2

Will the reducing conditions required for digestion impact the interchain disulfide bond integrity?

Felicia Mejare · Accepted Answer

Depending on what reducing agent and what concentration you use you can obtain intact Fab and not reduce the interchain disulfide bond. The light chain (LC) and heavy chain (HC) will not dissociate under native conditions due to hydrogen/hydrophobic forces. Before you analyze your samples with SDS-PAGE you must remove the cysteine otherwise the Fab will be reduced to Fd and LC during sample preparation for the SDS-PAGE. This can be done by a simple buffer exchange.

Question 3

I work with mouse IgG2a containing LC-lambda, can I use another affinity resin for purification of the Fab?

Felicia Mejare · Accepted Answer

Yes. There is the possibility to exchange the CaptureSelect LC-kappa (mur) columns to columns containing CaptureSelect LC-lambda (mouse). This resin is specific for mouse IgG. Please contact support@genovis.com if this is of interest.

Question 4

Can I digest Fc-fusion proteins using FabULOUS (SpeB)?

Felicia Mejare · Accepted Answer

Yes, FabULOUS should be able to digest a Fc fusion protein as long as you have not fused the protein too close to the cleavage site. FabULOUS digests human IgG1 just above the hinge (...KTHT / CPPCP...).

Question 5

Can I purify Fab fragments from human IgG with FabULOUS Fab kit?

Felicia Mejare · Accepted Answer

No, FabULOUS Fab kit contains CaptureSelect LC-kappa (mur) spin columns with selective affinity for murine LC-kappa. If you need to generate pure Fab from human IgG1 you can use either FabALACTICA Fab Kit or GingisKHAN Fab Kit. If you have a different subclass of human IgG, please contact support@genovis.com for guidance.

Question 6

Can I purify Fab fragments from human IgG with FabULOUS Fab kit?

Felicia Mejare · Accepted Answer

No, FabULOUS Fab kit contains CaptureSelect LC-kappa (mur) spin columns with selective affinity for murine LC-kappa. If you need to generate pure Fab from human IgG1 you can use either FabALACTICA Fab Kit or GingisKHAN Fab Kit. If you have a different subclass of human IgG, please contact support@genovis.com for guidance.

Question 7

There are not yet any FAQ's for this product

kk · Accepted Answer

If you have a technical question, please contact us.

Question 8

There are not yet any FAQ's for this product

kk · Accepted Answer

If you have a technical question, please contact us.

Question 9

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 10

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 11

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 12

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 13

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 14

There are not yet any FAQ's for this product

kk · Accepted Answer

If you have a technical question, please contact us.

Question 15

There are not yet any FAQ's for this product

kk · Accepted Answer

If you have a technical question, please contact us.

Question 16

Can FabRICATOR be stored in frozen stock solutions?

kk · Accepted Answer

We have no stability data for storage at -20°C or -70°C. However, we have customer reporting that they successfully stored reconstituted FabRICATOR at -20°C or -70°C for a couple of months. Genovis cannot guarantee the activity of the enzyme, but these storage conditions might be OK depending on the intended application.

Question 17

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 18

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 19

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 20

Can I use FabRICATOR and PNGaseF in the same reaction?

Felicia Mejare · Accepted Answer

Yes, PNGaseF and FabRICATOR can be used in a one-pot reaction as long as the reaction conditions follow the specifications for the FabRICATOR enzyme. It is important to have native reaction conditions for FabRICATOR to remain active.

Question 21

Can I use FabRICATOR and PNGaseF in the same reaction?

Felicia Mejare · Accepted Answer

Yes, PNGaseF and FabRICATOR can be used in a one-pot reaction as long as the reaction conditions follow the specifications for the FabRICATOR enzyme. It is important to have native reaction conditions for FabRICATOR to remain active.

Question 22

There are not yet any FAQ's for this product

kk · Accepted Answer

If you have a technical question, please contact us.

Question 23

There are not yet any FAQ's for this product

kk · Accepted Answer

If you have a technical question, please contact us.

Question 24

Will OglyZOR work on extended core 1 structures or single HexNAc?

kk · Accepted Answer

No, OglyZOR recognizes the core 1 disaccharide HexNAcHex (or GalNAcGal) and to some extend also the core 3 disaccharide HexNAc(2) (or GalNAcGlcNAc).

Question 25

Will OglyZOR leave the N-glycans intact?

kk · Accepted Answer

OglyZOR only works on desialylated O-glycans therefore is a sialidase (SialEXO) included with the product. SialEXO is not O-glycan specific and will therefore desialylate any N-glycans that are present.

Question 26

Can I use OglyZOR and PNGaseF in the same reaction?

kk · Accepted Answer

Yes, we have performed successful experiments with OglyZOR, SialEXO and PNGaseF in the same reaction under native conditions O/N at pH ~7.0.  If denaturing reaction conditions are required for PNGaseF, the reaction with OglyZOR and SialEXO needs to be performed first under native conditions, then add PNGaseF and the required buffer.

Question 27

Can OglyZOR be used to deglycosylate peptides generated with OpeRATOR?

kk · Accepted Answer

Yes, this workflow enables MS/MS of O-glycosylated peptides with removed O-glycans.

Question 28

Can OglyZOR be used to deglycosylate peptides generated with OpeRATOR?

kk · Accepted Answer

Yes, this workflow enables MS/MS of O-glycosylated peptides with removed O-glycans.

Question 29

How can I stop the GingisKHAN reaction?

kk · Accepted Answer

The enzyme is a cysteine protease that can be inhibited by alkylating agents such as iodoacetamide. There are also several other known inhibitors to this enzyme, like KYT-36.

Question 30

Can I use GingisKHAN on other subclasses than human IgG1?

Felicia Mejare · Accepted Answer

No. GingisKHAN is a protease that digests at exposed Lysines with no secondary structure restrictions. At the recommended reaction conditions, GingisKHAN digestion above the hinge has only been confirmed on human IgG1 with the exposed lysine above the hinge, (...KSCDK / THTCPPCP...). No digestion has been confirmed on other subclasses of human IgG except for several digestion sites in human IgG3.

Question 31

Is it possible to prepare Fab fragments from different antibodies using GingisKHAN Fab kit?

Felicia Mejare · Accepted Answer

Yes. GingisKHAN is provided lyophilized. After reconstitution, the enzyme can be used for up to 4 different antibodies. The digestion is performed in solution and you can prepare Fab fragments from 4 x 0.5 mg human IgG1. The purification step is done with Capture Select columns and 4 columns are provided in the kit.

Question 32

Is GingisKHAN an IgG specific protease?

kk · Accepted Answer

No, GingisKHAN is a general protease that with the recommended reaction conditions, 1 hour with light reducing conditions, digests human IgG1 at one specific site (...KSCDK/THTCPPCP...) above the hinge, generating Fab and Fc.

Question 33

Can I digest Fc-fusion proteins using GingisKHAN (Kgp)?

Felicia Mejare · Accepted Answer

Yes, GingisKHAN should be able to digest a Fc fusion protein if you have not fused the protein too close to the cleavage site and the Fc-part is from a human IgG1. GingisKHAN digests human IgG1 just above the hinge (...KSCDK / THTCPPCP...). If there are exposed Lysines in the fusion part, digestion might occur there as well.

Question 34

Can I digest Fc-fusion proteins using GingisKHAN (Kgp)?

Felicia Mejare · Accepted Answer

Yes, GingisKHAN should be able to digest a Fc fusion protein if you have not fused the protein too close to the cleavage site and the Fc-part is from a human IgG1. GingisKHAN digests human IgG1 just above the hinge (...KSCDK / THTCPPCP...). If there are exposed Lysines in the fusion part, digestion might occur there as well.

Question 35

Can I re-use FabRICATOR Z 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 36

Can I digest all mouse IgG subclasses with FabRICATOR Z?

Felicia Mejare · Accepted Answer

No, FabRICATOR Z only digests mouse IgG2a (….CPAPNLLG / GPSVF.. ) and IgG3 (…CPAPNILG / GPSVF…) at one specific site below the hinge. If Fab fragments are an option, we recommend our FabULOUS enzyme that digest mouse IgG1 above the hinge. FabULOUS requires light reducing conditions for optimal activity. If strong reducing conditions are used, this may likely cause reduction of the interchain disulfides.

Question 37

Can I digest rat IgG2a with FabRICATOR Z?

Felicia Mejare · Accepted Answer

Unfortunately, FabRICATOR Z does not digest rat IgG2a. If generating Fab fragments is an option, we suggest using FabULOUS enzyme that digests rat IgG2a. FabULOUS does not have a single specific cleavage site, but a primary digestion site, so there can be more than one digestion site. The digestion site(s) for rat IgG2a using FabULOUS is not determined, but the generated fragments are Fab and Fc.

Question 38

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 39

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.

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FabULOUS

GalNAcEXO

GalactEXO

FucosEXO

FabRICATOR Validation Kit

GlySERIAS

OglyZOR

Antibody LC-MS Analysis

GingisKHAN

FabRICATOR Z