€625.00
FabULOUS (SpeB) digests IgG from several species in the hinge under reducing conditions
FabULOUS (SpeB) is a cysteine protease that digests IgG from several different species and subclasses in the hinge region generating Fc and Fab fragments. FabULOUS digests human IgG1 at a specific site above the hinge while IgG from other species and subclasses such as mouse IgG1 is digested in the hinge region. FabULOUS requires reducing conditions and is available as lyophilized powder for digestion of 2 mg of IgG.
| Application | |
|---|---|
| Product Group | |
| Source/Origin | Cloned from S. pyogenes, Produced in E. coli (His-tagged), 28.7 kDa |
| Product Composition | |
| Digestion Site | |
| Specificity | |
| Substrates | Fc-fusion proteins, Goat IgG, Human IgG1, Human IgG2, Human IgG3, Human IgG4, Mouse IgG, Rabbit IgG, Rat IgG, Sheep IgG |
| Reaction Conditions | 1 hour reaction at 37°C according to protocol specifications |
| Digestion Buffers | PBS (pH 6.5 – 8.0), TBS (pH 7.0 – 8.0), Sodium Acetate (pH 6.0) |
| Buffer Compatibility | Below pH 6.5, the enzyme concentration needs to be increased for efficient digestion of IgG. |
| Additives/Co-factors | Requires Reducing Conditions, (Cysteine, Cysteamine, 2-Mercaptoethanol, DTT, TCEP) |
| 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.
