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.

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.

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.

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.

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.