Does IgMBRAZOR digest only human IgM?

IgM from some species of monkey may also be digested by IgMBRAZOR.

Can IgMBRAZOR and FabRICATOR be used in the same reaction?

Yes, IgMBRAZOR and FabRICATOR can be used in combination.

Can IgMBRAZOR and PNGase F be used in the same reaction?

Yes, IgMBRAZOR and PNGase F can be used in combination if the reaction is performed under non-denaturing conditions. However, for complete deglycosylation of IgM using PNGase F denaturing conditions may be required and it is then recommended to perform the IgMBRAZOR digestion prior the PNGase F deglycosylation.

Is it possible to use IgMBRAZOR with very low concentrations of IgM?

It is possible to use IgMBRAZOR with low concentrations of IgM. However, this will lead to a decreased reaction rate, so for IgM concentrations below 0.5 mg/mL it is recommended to increase the amount of added enzyme and/or increase the incubation time. There is no risk of overdigestion and the incubation time can be prolonged to overnight if necessary.

Which buffers are compatible with IgMBRAZOR?

The enzyme is active at pH 5.5 to 9.0, tolerates up to 300 mM NaCl, and does not require reducing conditions or co-factors for activity. PBS and TBS buffers are both compatible. Optimization may be required if buffers other than the recommended are used.

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 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 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