Frequently Asked Questions
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Which digestion buffers are compatible with FabRICATOR HPLC?
We have tested PBS and 150 mM ammonium acetate, pH7 with good results. More buffers were found to be compatible with FabRICATOR digestion in solution (more info) and should be suitable for FabRICATOR HPLC as well. However, the ionic strength of the buffer influences retention of the antibody fragments on the FabRICATOR HPLC column and a minimum of 100-150 mM salt is necessary for IgG1 subunits to elute as a single, narrow peak. Other IgG subclasses or fusion proteins might need some optimization of buffer composition.
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How can I stop the FabULOUS reaction?
FabULOUS enzyme reaction is inhibited with iodoacetamide, iodo acetic acid or lowering pH below 3.
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Can I re-use FabRICATOR 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.
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Does FabULOUS need reducing condition to work?
Yes, FabULOUS requires reduced condition for activity and reaction is performed in the presence of 1-5 mM DTT or TCEP at neutral pH.
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Is FabULOUS working on human IgG4?
Yes, but a longer incubation time is needed.
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What is the FabRICATOR MagIC?
The FabRICATOR MagIC magnetic beads with immobilized FabRICATOR (IdeS) enzyme.
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Which digestion buffers are compatible with FabRICATOR MagIC?
The protocol for FabRICATOR MagIC is based on using standard PBS pH 7.4 as digestion buffer. FabRICATOR in solution is compatible with most commonly used buffers at pH=6-8 and the same buffers should work for FabRICATOR MagIC, but the incubation time might need to be optimized.
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Can I use FabRICATOR MagIC in a automation system?
FabRICATOR MagIC is suitable for using in automated systems that can handle magnetic beads. On the applications page you can read about how it’s used in a KingFisher™ workflow.
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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.
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Can you combine GalactEXO with SialEXO or SialEXO 23 to remove both galactoses and sialic acids in a single reaction?
Yes, you can. All three enzymes perform well in the same reaction buffer (20 mM Tris pH 6.8). There is no need to increase incubation time when combining the enzymes.
Learn more about GalactEXO®