Quickly and easily generate and purify Fab fragments from IgG.

The Fab Preparation Kits provide for simple generation and purification of antibody Fab fragments from whole IgG. 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.

The 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.

These Fab Preparation Kits are suitable for human, rabbit, mouse and other species and subclasses of IgG. For best results with mouse IgG1, use the ficin-based kit, Product # 44980.

Figure 1. Digestion and purification scheme for preparing Fab fragments from IgG antibodies.

Why make Fab Fragments?

Because of their smaller size as functional components of the whole molecule, antibody fragments offer several advantages over intact antibodies for use in certain immunochemical techniques and experimental applications. Some of the possible advantages of Fab fragments include...

Reduced nonspecific binding that results from Fc interactions (many cells have receptors that bind the Fc region) Ability to control binding to Protein A in experiments involving immunoprecipitation and Western blotting More efficient penetration of tissue sections, resulting in improved staining for immunohistochemical applications Potentially higher sensitivity in antigen detection in solid phase applications as a result of reduced steric hindrance from large protein epitopes Elimination of Fc-associated effector functions (e.g,. complement fixation) in antigen-antibody binding studies Simpler system for study the structural basis for immune recognition using X-ray cyrstallography or NMR Lower immunogenicity that intact antibody, an advantage for experiments in vivo

Table 1. Recommended digestion times for various species and concentrations of IgG. Species [IgG] (mg/ml) Digestion Time (hours) Rabbit    8 8-9 4 6-7 1.5 4-5 0.5 3-4 Human    8 5-6 4 5-6 1.5 3-4 0.5 2-3 Mouse    8 4-5 4 3-4 1.5 2-3 0.5 2-3

Highlights

• 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, but papain-based digestion is effective for many species and subclasses of IgG including rat, goat and pig (*Note: for best results with mouse IgG1, use Product # 44980)
• High capacity – kit provides for digestion and purification from as much as 4 mg of IgG at a time. Use the standard size kit for fragmenting 0.25 to 4 mg IgG, and the micro kit for 25 to 250 ug IgG.
• 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

Figure 2. Typical SDS-PAGE (10% Bis-Tris) result of reduced and non-reduced Fab and Fc fragments from rabbit IgG.

Related Products
F(ab)2 Preparation Kit
Mouse IgG1 Fab and F(ab)2 Preparation Kit
IgM Fragmentation Kit

Product #	Description	Pkg. Size
44685	Pierce Fab Micro Preparation Kit Makes and purifies Fab fragments from ten antibody samples containing 25-250 µg IgG. Kit contains: Immobilized Papain, 0.5 ml Cysteine-HCl, 1 g Fab Digestion Buffer, 55 ml NAb Protein A Spin Columns, 2 x 0.2 ml Phosphate Buffered Saline, 2 packs IgG Elution Buffer, 50 ml Zeba Desalt Spin Columns, 10 x 0.5 ml Spin Columns and Accessories	Kit
44985	Pierce Fab Preparation Kit Makes and purifies Fab fragments from ten antibody samples containing 0.25-4 mg IgG. Kit contains: Immobilized Papain, 1.25 ml Cysteine-HCl, 1 g Fab Digestion Buffer, 120 ml NAb Protein A Spin Column, 1 ml Phosphate Buffered Saline, 2 packs IgG Elution Buffer, 120 ml Zeba Desalt Spin Columns, 10 x 2 ml Spin Columns and Accessories	Kit