FabULOUS Fab kit contains FabULOUS (SpeB) enzyme and CaptureSelect™ spin columns for affinity capture of murine LC-kappa. FabULOUS digests mouse IgG in the hinge region generating Fc and Fab fragments. CaptureSelect™ spin columns capture murine LC-kappa for purification of Fab. FabULOUS Fab kit is available in spin column format for digestion and purification of 2mg of mouse IgG.
| Application | |
|---|---|
| Product Group | |
| Source/Origin | Cloned from S. pyogenes, Produced in E. coli (His-tagged), 28.7 kDa |
| Product Composition | Lyophilized in TBSl, CaptureSelect LC-kappa (mur) columns included |
| Digestion Site | |
| Specificity | |
| Substrates | |
| Reaction Conditions | 1 hour reaction at 37°C according to protocol specifications |
| Digestion Buffers | |
| Buffer Compatibility | Digestion time may need to be optimized for individual antibodies. |
| Additives/Co-factors | Requires Reducing Conditions, 30-50 mM Cys is enough for activity and should not be exceeded when intact Fab fragments are required., Use of the DTT and TCEP will not yield intact Fab fragments. |
| Inhibitors | |
| Unit Definition |
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…).
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.
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
Yes, FabULOUS requires reduced condition for activity and reaction is performed in the presence of 1-5 mM DTT or TCEP at neutral pH.
FabULOUS enzyme reaction is inhibited with iodoacetamide, iodo acetic acid or lowering pH below 3.
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.
Yes, but a longer incubation time is needed.
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.
