Thermo Scientific Pierce Fab Preparation Kit uses immobilized papain protease to digest human or mouse IgG antibodies to make separate Fab and Fc fragments and subsequently to purify the Fab using Protein A agarose.
Features of the Fab Preparation Kit:
• High capacity—use the standard size kit for fragmenting 0.25 to 4 mg IgG. • Enzyme-free digestion products—Immobilized Papain (beaded agarose resin) provides for control of the digestion reaction and complete removal of resulting antibody fragments from the proteolytic enzyme • Suitable for human and other species of IgG—the kit procedure is optimized for human, mouse and rabbit IgG. Papain-based digestion is effective for certain other species and subclasses of IgG (e.g., goat and pig), although the purification step requires that the antibody effectively binds to Protein A. (Note: for best results with mouse IgG1, use Part No. 44980.) • Provides ready-to-use Fab—digestion and final recovery of purified Fab fragments occurs in neutral pH sodium phosphate buffer, suitable for storage or immediate use in typical applications • Complete—kits include all reagents needed to prepare and purify antibody fragments • Fast—spin format greatly reduces sample processing time • Flexible—Protocols are included for multiple species and IgG subclasses, as well as sample size and concentration. • Efficient—enhanced yield and sample purity
These Fab Preparation Kits are suitable for human, rabbit, mouse and other species and subclasses of IgG. The papain antibody digestion reaction is performed in convenient disposable spin columns that allow efficient removal of the immobilized protease and maximum recovery of the IgG fragments. Also included in the kits are Thermo Scientific NAb Protein A Spin Columns and buffers to efficiently purify the resulting fragments. Protein A binds the Fc fragments and undigested IgG, allowing the pure Fab fragments to be recovered in the flow-through fraction. The kits also include Thermo Scientific Zeba Desalting Spin Columns for preparing the IgG sample quickly without dilution instead of utilizing time-consuming dialysis steps.
The kits use papain, a nonspecific thiol-endopeptidase, to enzymatically cleave whole IgG just above the hinge region to create two separate Fab fragments and one Fc fragment per antibody molecule. Because the papain protease is supplied in immobilized form as beaded agarose resin, the digestion reaction is easily stopped by removing the resin from the IgG solution; the result is digest products that are enzyme-free.