Frequently Asked Questions

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Can I use FabRICATOR (IdeS) with very low amounts of IgG?

It is possible to use FabRICATOR with low concentrations of IgG. Low concentrations of IgG will lead to decreased reaction rate, so for concentrations below 0.5mg/mL it is therefore recommended to increase the amount of added enzyme and/or increase the incubation time. There is no risk of over-digestion and the incubation time can be prolonged to O/N if necessary.

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

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.

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Can FabRICATOR be stored in frozen stock solutions?

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.

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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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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.

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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.

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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: ..CPAPELLG / GPSVF..
Human IgG2: ..CPAPPVA / GPSVF..
Human IgG3: ..CPAPELLG / GPSVF..
Human IgG4: ..CPAPEFLG / GPSVF..

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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.

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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.

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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.

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