Fab with FabULOUS

Fab Antibody Fragment Generation with FabULOUS

Intact antibodies can be dissected into smaller fragments to isolate certain characteristic properties of the antibody. The generated fragments can be used for subunit analysis or in binding studies where properties associated with a specific fragment are unwanted. Enzymatic proteolysis is a rapid method to produce antibody fragments, but challenges are often associated with unspecific digestion and reducing conditions. FabULOUS digests IgG from several species and subclasses in the hinge under mild reducing conditions to generate intact Fab and Fc fragments.

FabULOUS workflow - Mouse Fab

Generating Intact Fab and Fc Fragments

The preparation of intact antibody fragments from many different species and subclasses of IgG can be challenging due to unspecific enzymatic activity and the reducing reaction conditions often required that may reduce the interchain disulfide bonds.  FabULOUS is a cysteine protease that digests in the hinge region of IgG and is a valuable research tool that can generate intact Fab fragments from several species of IgG including human, mouse, rat, goat, sheep and rabbit. FabULOUS is active under mild reducing conditions that allows for the generation of intact Fab fragments without reduction of interchain disulfides (Fig. 1). The reaction is performed at neutral pH with a mild reducing agent and the digestion complete after incubation at 37 °C for 1 hour.

 

Human & Mouse Fab

Figure 1.SDS-PAGE analysis of human IgG1 and IgG2 and mouse IgG1 and IgG2a digested in the hinge region using FabULOUS. 

 

 

 

Intact Fab from Mouse IgG with FabULOUS® Fab kit

For the generation of intact and pure mouse IgG Fab fragments without residual enzyme in the final preparation, the FabULOUS enzyme is combined with affinity purification in the FabULOUS Fab kit. With this kit, FabULOUS digestion of the mouse IgG is followed by separation of the Fab fragments from the FabULOUS enzyme and Fc fragments using the CaptureSelect spin column with affinity resin towards murine LC kappa chain. The enzyme and Fc portion of the sample is found in the flow-through of the column and the Fab part is after wash of the resin eluted by lowering the pH (Fig. 2). Using the FabULOUS Fab kit, Fab fragments from up to 2 mg of mouse IgG can be easily prepared.

Figure 2. SDS-PAGE under non-reducing conditions of mouse IgG1 digested by FabULOUS.

  1. Molecular weight marker
  2. Intact mouse IgG1
  3. Fab and Fc generated by FabULOUS
  4. Flow-through of Fc and enzyme
  5. Eluted Fab fragments


 

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