Quickly and easily generate and purify F(ab')2 fragments from IgG.

The F(ab')2 Preparation Kits provide for efficient generation and purification of antibody F(ab')2 fragments from whole IgG. The kits use pepsin, a nonspecific endopeptidase, to enzymatically digest the Fc portion of whole IgG to yield the fragment known as F(ab')2. This fragment is composed of a pair of Fab' units connected by two disulfide bonds. Because the pepsin 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 F(ab')2 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 F(ab')2 Preparation Kits are suitable for human, rabbit, mouse and other species and subclasses of IgG except mouse IgG1 which pepsin cleaves poorly. (To prepare F(ab')2 fragments from mouse IgG1, use the ficin-based kit, Product # 44980).

Figure 1. Digestion and purification scheme for preparing F(ab')2 fragments from IgG antibodies.

Why make F(ab')2 Fragments?

Because of their smaller size as functional components of the whole molecule, Fab and F(ab')2 antibody fragments offer several advantages over intact antibodies for use in certain immunochemical techniques and experimental applications. Fab fragments are the smallest antigen-binding fragments, but have limited utility because of their monovalent structure. F(ab')2 fragments derive particular utility from having no Fc domain and somewhat smaller size than whole IgG while also maintaining avidity and other properties of divalent structure. Some general advantages of Fab and F(ab')2 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

Highlights

• Enzyme-free digestion products – Immobilized Pepsin (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, rabbit and mouse* IgG, but pepsin-based digestion is effective for other species and subclasses of IgG (*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.
• 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

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

References

1. Smith-Jones, P.M., et al. (2004). Nature Biotech. 22: 32-37.
2. Pruimboom, I.M., et al. (1999). Infect. Immun. 67(3): 1292-1296.
3. Wedrychowski, A., Kim, Y., and Chang, T.W. (1993) Immune enhancers composed of polyvalent binding sites of anti-CD3 antibodies. BioTechnology 11: 486.
4. Lamoyi, E. (1986). Preparation of F(ab')2 fragments from mouse IgG of various subclasses. Meth. Enzymol. 121: 652-663.
5. Kulkarni, P.N., et al. (1985). Conjugation of methotrexate to IgG antibodies and their F(ab')2 fragments and the effect of conjugated methotrexate on tumor growth in vivo. Cancer, Immunol., Immunotherapy 19: 211-214.
6. Rousseaux, J., et al. (1983). Optimal conditions for the preparation of Fab and F(ab')2 fragments from monoclonal IgG of different rat IgG subclasses. J. Immunol. Meth. 64: 141-146.