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

Is it possible to prepare Fab fragments from different antibodies using GingisKHAN Fab kit?

Yes. GingisKHAN is provided lyophilized. After reconstitution, the enzyme can be used for up to 4 different antibodies. The digestion is performed in solution and you can prepare Fab fragments from 4 x 0.5 mg human IgG1. The purification step is done with Capture Select columns and 4 columns are provided in the kit.

Can I digest Fc-fusion proteins using GingisKHAN (Kgp)?

Yes, GingisKHAN should be able to digest a Fc fusion protein if you have not fused the protein too close to the cleavage site and the Fc-part is from a human IgG1. GingisKHAN digests human IgG1 just above the hinge (…KSCDK / THTCPPCP…). If there are exposed Lysines in the fusion part, digestion might occur there as well.

Can I use GingisKHAN on other subclasses than human IgG1?

No. GingisKHAN is a protease that digests at exposed Lysines with no secondary structure restrictions. At the recommended reaction conditions, GingisKHAN digestion above the hinge has only been confirmed on human IgG1 with the exposed lysine above the hinge, (…KSCDK / THTCPPCP…). No digestion has been confirmed on other subclasses of human IgG except for several digestion sites in human IgG3.

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 immobilized FabALACTICA 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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