Submit a Support Case

Genovis_Webb_Fragetecken-1

FAQ

Here you can find all FAQ we have on Genovis products. Don't hesitate to contact us if you can't find an answer to your question.

FabRICATOR


There should not be any problems to use low concentrations of IgG. If you don't get complete digestion, try to increase the incubation time. There is no risk of over digestion and the incubation time can be prolonged to over night if necessary.

An alternative would be to increase the ratio enzyme to antibody (i.e. Follow protocol for mouse IgG). We have not made any specific tests on low concentrations but we have not heard that it would be any problems.


FabRICATOR is very specific and small changes in the sequence (..CPAPELLGGPSVF..) may reduce or completely impair the ability to digest the IgG.

We have tested a few different modified sequences and we can see that the enzyme digest the IgG to some degree to Fab2 and Fc under somewhat modified reaction conditions. We then obtained a mixture of Fc, Fab2 and single cut mAb (one of the HC still undigested).

We used PBS buffer but with greatly reduced salt concentration (5-10 mM instead of 140 mM) which appeared to promote digestion. We also added 10X more enzyme than recommended amount and prolonged incubation time.

It is very difficult to predict the outcome for a specific modification. It might work, but we cannot guarantee it unfortunately.


Yes, FabRICATOR works on hamster IgG using the standard protocol as for human IgG.


Yes, FabRICATOR can cleave Fc-Fusion proteins. For example Enbrel is effectivelty cleaved by FabRICATOR into Fc and TNFR. Enbrel (etanercept) is a fusion protein composed of the Fc domain of hIgG1 and the p75 tumor necrosis factor receptor (TNFR).


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


If we assume that the antibody concentration in serum is 15 mg/ml you would have to add 10-15 000 units to a sample of 1 ml serum. In 50 µl serum you would have to add 500 - 750 units of FabRICATOR®.


FabRICATOR® cleave the IgG molecule in one specific site in the lower hinge region of the anitbody leaving a F(ab')2 fragment and a Fc-fragment.

Human IgG1: HTCPPCPAPELLG⬇GPSVF

Human IgG2: VECPPCPAPP_VA⬇GPSVF

Human IgG3: PPCPRCPAPELLG⬇GPSVF

Human IgG4: PHAHHAQAPEFLG⬇GPSVF


According to the reference listed below, FabRICATOR does not digest IgM.

2002 paper by von Pavel-Rammingen et al titled "IdeS, a novel streptococcal cysteine proteinase with uniqe specificity for immunoglobulin G"


FabRICATOR belongs to the cysteine protease family. There should be no modification other than the cleavage of the peptide bond between the two glycines. The c-terminal will regain the carboy 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


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.


We have not tested storing the enzymen in glycerol solutions. However, many of our customers have successfully stored the enzyme (after it was reconstituted with water) at both -70°C and -20°C for a few months.

FragIT & FragIT Kit

FabRICATOR® cleave the IgG molecule in one specific site in the lower hinge region of the anitbody leaving a F(ab')2 fragment and a Fc-fragment.Human IgG1: HTCPPCPAPELLG⬇GPSVF

Human IgG2: VECPPCPAPP_VA⬇GPSVF

Human IgG3: PPCPRCPAPELLG⬇GPSVF

Human IgG4: PHAHHAQAPEFLG⬇GPSVF

You can indeed elute the Fc fragments from the CaptureSelect column using a low pH buffer commonly used for example during protein A/G purification of antibodies. We have successfully used 0.1 M glycine buffer pH 2.6-2.8 to release the Fc fragments. Be sure to adjust pH back to normal using a 1 M Tris-HCl buffer or similar directly after isolation of the Fc fragments from the CaptureSelect column.
To use protein A or MabSelect Sure (GE) for removal of Fc as a platform after digestion with FabRICATOR or FragIT is problematic. Therefore we have chosen CaptureSelect in the FragIT Kit for removal of Fc fragments. CaptureSelect is much more specific for Fc. Both protein A and MabSelect Sure (GE) may have affinity for Fab and the degree of affinity is antibody dependent.We have many customer reports on this subject and they usually comes back to us for the CaptureSelect columns for the above reason.

We can only guarantee optimal digestion for one time use. However, depending on antibody the columns can be reused but we do not have a proper sanitization protocol. We don't recommend that you run different antibodies on the same column as we cannot guarantee that there will be no carry-over. If desired to reuse the column store in 10-20% ethanol at +4-8°C.

IgGZERO & deGlycIT


First you could increase incubation time and increase temperature to 37°C.

Another explanation for not complete deglycosylation could be if the antibody has higher amounts of high-mannose, some hybrid glycans or bisected glycans. For these glycans IgGZERO has low or no activity. Especially, high-mannose glycans are particular resistant to digestion.

If longer incubation time and increased temperature does not increase deglycsylation we would recommend you to try our other endoglycosidase GlycINATOR (EndoS2). This enzyme has much higher activity on these types of glycans sometimes seen in m.Abs.


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

All FAQ


FabRICATOR belongs to the cysteine protease family. There should be no modification other than the cleavage of the peptide bond between the two glycines. The c-terminal will regain the carboy 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


You can indeed elute the Fc fragments from the CaptureSelect column using a low pH buffer commonly used for example during protein A/G purification of antibodies. We have successfully used 0.1 M glycine buffer pH 2.6-2.8 to release the Fc fragments. Be sure to adjust pH back to normal using a 1 M Tris-HCl buffer or similar directly after isolation of the Fc fragments from the CaptureSelect column.


FabULOUS enzyme reaction is inhibited with iodoacetamide, iodo acetic acid or lowering pH below 3.


To use protein A or MabSelect Sure (GE) for removal of Fc as a platform after digestion with FabRICATOR or FragIT is problematic. Therefore we have chosen CaptureSelect in the FragIT Kit for removal of Fc fragments. CaptureSelect is much more specific for Fc. Both protein A and MabSelect Sure (GE) may have affinity for Fab and the degree of affinity is antibody dependent.

We have many customer reports on this subject and they usually comes back to us for the CaptureSelect columns for the above reason.


Yes, columns can be reused but make sure you wash the column with buffer. It can be stored in 20% ethanol as well as buffer.  

However, we do not have a proper sanitization protocol so we don't recommend that you run different antibodies on the same column as we cannot guarantee that there will be no carry-over. From an enzymatic activity point of view the column will work for several runs but eventually enzymatic activity will decline.


We have not tested storing the enzymen in glycerol solutions. However, many of our customers have successfully stored the enzyme (after it was reconstituted with water) at both -70°C and -20°C for a few months.


Yes, FabULOUS requires reduced condition for activity and reaction is performed in the presence of 1-5 mM DTT or TCEP at neutral pH.


FabRICATOR is very specific and small changes in the sequence (..CPAPELLGGPSVF..) may reduce or completely impair the ability to digest the IgG.

We have tested a few different modified sequences and we can see that the enzyme digest the IgG to some degree to Fab2 and Fc under somewhat modified reaction conditions. We then obtained a mixture of Fc, Fab2 and single cut mAb (one of the HC still undigested).

We used PBS buffer but with greatly reduced salt concentration (5-10 mM instead of 140 mM) which appeared to promote digestion. We also added 10X more enzyme than recommended amount and prolonged incubation time.

It is very difficult to predict the outcome for a specific modification. It might work, but we cannot guarantee it unfortunately.


First you could increase incubation time and increase temperature to 37°C.

Another explanation for not complete deglycosylation could be if the antibody has higher amounts of high-mannose, some hybrid glycans or bisected glycans. For these glycans IgGZERO has low or no activity. Especially, high-mannose glycans are particular resistant to digestion.

If longer incubation time and increased temperature does not increase deglycsylation we would recommend you to try our other endoglycosidase GlycINATOR (EndoS2). This enzyme has much higher activity on these types of glycans sometimes seen in m.Abs.


Yes, FabRICATOR works on hamster IgG using the standard protocol as for human IgG.


There should not be any problems to use low concentrations of IgG. If you don't get complete digestion, try to increase the incubation time. There is no risk of over digestion and the incubation time can be prolonged to over night if necessary.

An alternative would be to increase the ratio enzyme to antibody (i.e. Follow protocol for mouse IgG). We have not made any specific tests on low concentrations but we have not heard that it would be any problems.


We have not found that FabULOUS (SpeB) digest human IgG4. For other subclasses and species tested please visit www.genovis.com/applications


According to the reference listed below, FabRICATOR does not digest IgM.

2002 paper by von Pavel-Rammingen et al titled "IdeS, a novel streptococcal cysteine proteinase with uniqe specificity for immunoglobulin G"


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.


FabRICATOR® cleave the IgG molecule in one specific site in the lower hinge region of the anitbody leaving a F(ab')2 fragment and a Fc-fragment.

Human IgG1: HTCPPCPAPELLG⬇GPSVF

Human IgG2: VECPPCPAPP_VA⬇GPSVF

Human IgG3: PPCPRCPAPELLG⬇GPSVF

Human IgG4: PHAHHAQAPEFLG⬇GPSVF


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


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


If we assume that the antibody concentration in serum is 15 mg/ml you would have to add 10-15 000 units to a sample of 1 ml serum. In 50 µl serum you would have to add 500 - 750 units of FabRICATOR®.


Yes, FabRICATOR can cleave Fc-Fusion proteins. For example Enbrel is effectivelty cleaved by FabRICATOR into Fc and TNFR. Enbrel (etanercept) is a fusion protein composed of the Fc domain of hIgG1 and the p75 tumor necrosis factor receptor (TNFR).