I work with mouse IgG2a containing LC-lambda, can I use another affinity resin for purification of the Fab?
Yes. There is the possibility to exchange the CaptureSelect LC-kappa (mur) columns to columns containing CaptureSelect LC-lambda (mouse). This resin is specific for mouse IgG. Please contact support@genovis.com if this is of interest.
Can I purify Fab fragments from human IgG with FabULOUS Fab kit?
No, FabULOUS Fab kit contains CaptureSelect LC-kappa (mur) spin columns with selective affinity for murine LC-kappa. If you need to generate pure Fab from human IgG1 you can use either FabALACTICA Fab Kit or GingisKHAN Fab Kit. If you have a different subclass of human IgG, please contact support@genovis.com for guidance.
Can I digest Fc-fusion proteins using FabULOUS (SpeB)?
Yes, FabULOUS should be able to digest a Fc fusion protein as long as you have not fused the protein too close to the cleavage site. FabULOUS digests human IgG1 just above the hinge (…KTHT / CPPCP…).
Will the reducing conditions required for digestion impact the interchain disulfide bond integrity?
Depending on what reducing agent and what concentration you use you can obtain intact Fab and not reduce the interchain disulfide bond. The light chain (LC) and heavy chain (HC) will not dissociate under native conditions due to hydrogen/hydrophobic forces. Before you analyze your samples with SDS-PAGE you must remove the cysteine otherwise the Fab will be reduced to Fd and LC during sample preparation for the SDS-PAGE. This can be done by a simple buffer exchange.
Does FabULOUS digest all human IgG subclasses above the hinge?
FabULOUS is a cysteine protease that digests IgG in the hinge region. A primary digestion site for human IgG1 is above the hinge (…KTHT / CPPCP…). The enzyme is also active on human IgG2 and IgG3, digestion site not determined but Fab and Fc is generated. FabULOUS digests human IgG4 primarily at a site below the hinge using significantly longer incubation, generating Fab’ due to the reducing conditions. The possibility of other, secondary digestions sites is antibody dependent
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.
How can I stop the FabULOUS reaction?
FabULOUS enzyme reaction is inhibited with iodoacetamide, iodo acetic acid or lowering pH below 3.