Can I re-use FabALACTICA Immobilized columns?
We can only guarantee optimal digestion for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.
Can I digest Fc-fusion proteins using FabALACTICA (IgdE)?
No, FabALACTICA has shown very poor activity on Fc-fusion proteins.
How can I remove residual enzyme from the digested antibody in solution?
We recommend using FabALACTICA Immobilized for digesting human IgG1 and generating a final preparation free of any residual enzyme. You can also use FabALACTICA Fab kit for digestion of human IgG1 and purification of the generated Fab fragments.
What is the difference between FabALACTICA and GingisKHAN?
FabALACTICA (IgdE) and GingisKHAN (Kgp) are both cysteine proteases that digest IgG1 above the hinge to generate intact Fab and Fc fragments. FabALACTICA is specific for human IgG1 and digests at a specific site (…KSCDKT / HTCPPCP…) with overnight incubation without the need for reducing conditions. GingisKHAN is a general protease that with the recommended reaction conditions, 1 hour with light reducing conditions, digests human IgG1 at a specific site (…KSCDK/THTCPPCP…).
Can I re-use FabRICATOR Z Immobilized columns?
We can only guarantee optimal digestion for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.
Can I digest rat IgG2a with FabRICATOR Z?
Unfortunately, FabRICATOR Z does not digest rat IgG2a. If generating Fab fragments is an option, we suggest using FabULOUS enzyme that digests rat IgG2a. FabULOUS does not have a single specific cleavage site, but a primary digestion site, so there can be more than one digestion site. The digestion site(s) for rat IgG2a using FabULOUS is not determined, but the generated fragments are Fab and Fc.
Can I digest all mouse IgG subclasses with FabRICATOR Z?
No, FabRICATOR Z only digests mouse IgG2a (….CPAPNLLG / GPSVF.. ) and IgG3 (…CPAPNILG / GPSVF…) at one specific site below the hinge. If Fab fragments are an option, we recommend our FabULOUS enzyme that digest mouse IgG1 above the hinge. FabULOUS requires light reducing conditions for optimal activity. If strong reducing conditions are used, this may likely cause reduction of the interchain disulfides.
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 Fab2 Kit for generation and purification of fragments from Rabbit IgG?
FabRICATOR Fab2 Kit digests rabbit IgG but to capture the Fc fragments in the affinity step you need another resin than the CaptureSelect™ Fc normally included in the kit. Please contact the Genovis team if you are interested in processing rabbit IgG and we will provide columns with resin compatible with rabbit Fc in the FabRICATOR Fab2 Kit.
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.