Can I release Fc fragments from the CaptureSelect column after isolating the Fab fragments?
It is not recommended to elute the Fc fragments from the CaptureSelect column since the fraction might also include undigested or semi digested material.
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 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.
We want to make Fab’s directly from antibodies. When would the selection of FabALACTICA be the first choice over GingisKHAN or FabULOUS?
FabALACTICA does not require reducing conditions as both GingisKHAN and FabULOUS does, and allows for generation of Fab fragments from native human IgG1.