Preparation of F(ab’)2 and Fc Fragments using FabRICATOR®

Performance Activity

Generation and purification of F(ab’)2 and scFc fragments without residual enzyme, enabling rapid preparation of antibody fragments for downstream applications.

Some downstream applications such as binding studies are highly sensitive and, therefore, require pure antibody fragments without residual enzyme in the final preparation. Our IgG specific proteases are therefore also available in immobilized formats for fast and easy generation of homogenous F(ab’)2 and scFc fragments.

The FabRICATOR Immobilized contains immobilized FabRICATOR (Ides) to prepare F(ab’)2 and Fc fragments from several species of IgG (for rabbit IgG, please inquire), while mouse IgG2a and IgG3 can be processed using the FabRICATOR Z Fab2 Kit containing FabRICATOR Z Immobilized. To generate and purify intact F(ab’)2 and scFc fragments, trastuzumab was processed using the FabRICATOR Fab2 Kit and analyzed by SDS-page. First, the antibody was digested on the FabRICATOR Immobilized spin column at RT for 15 minutes followed by a short centrifugation step.

Robust digestion using FabRICATOR Immobilized

The F(ab’)2 fragments were purified on the CaptureSelect™ spin column and the bound Fc fragments eluted in a single step by lowering the pH followed by immediate adjustment back to neutral pH. The procedure from digestion to collection of fragments using FabRICATOR Fab2 Kit takes about one hour.

Purification of F(ab’)2 and Fc fragments following affinity purification

Generation and SDS-PAGE analysis of antibody fragments using FabRICATOR Fab2 Kit. Schematic representation of the FabRICATOR Fab2 Kit sample preparation (left). Non-reducing SDS-PAGE analysis of antibody framents (right); Lane 1) kDa markers, Lane 2) intact intact antibody, Lane 3) Fab'2 and Fc fragment after FabRICATOR Immobilized digestion, Lane 4) F(ab')2 in flow-through over Fc affinity capture column, Lane 5) Eluted Fc fragment.

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