Can FabALACTICA and FabRICATOR be used in the same reaction?

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.

Can I use FabRICATOR and PNGaseF in the same reaction?

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.

Does FabRICATOR (IdeS) digest hamster IgG?

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

Can I cleave Fc-fusion proteins with FabRICATOR?

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.

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

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

How much of the FabRICATOR do you need to cleave IgG in 50 µl of normal human serum?

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.

Where does FabRICATOR cleave the IgG molecule?

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

Does FabRICATOR (IdeS) have protease activity on IgM?

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.

Does FabRICATOR introduce modifications?

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.

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

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.

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