Can guanidinium hydrochloride be used as a denaturing agent instead of urea?

No, GingisREX is inhibited by GdHCl even in low concentrations. This chaotrope should be avoided.

Is the reaction ready in only 60 minutes?

The digestion time is affected by many factors like the enzyme to sample ratio, pH of the digestion reaction, and the sample characteristics. The reaction time needs to be optimized for each specific case.

Is cysteine really needed for activation of the cysteine protease if TCEP or DTT is added in the reaction?

Yes, cysteine is required for both activity and specificity of the GingisREX enzyme.

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