Life Science ResearchLife Science Research ›  Protein BiologyProtein Biology ›  Antibody Production and PurificationAntibody Production and Purification ›  Antibody Fragmentation KitsAntibody Fragmentation Kits › Pierce* Fab Preparation Kits
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ThermoScientific
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Pierce* Fab Preparation Kits

Thermo Scientific* Pierce Fab Preparation Kits use 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.
 
 
 
 
 
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 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. Thermo Scientific Zeba* Desalting Spin Columns make IgG-sample preparation quick and easy.

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 protease
  • 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 Part No. 44980)
  • High capacity – kit provides for digestion and purification from as much as 4mg of IgG at a time. Use the standard size kit for fragmenting 0.25 to 4mg IgG, and the micro kit for 25 to 250µg 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 and easy to use – kits include all reagents, spin columns and accessories needed to prepare and purify antibody fragments
Includes: Papain agarose resin, cysteine, digestion buffer, Protein A columns with binding and elution buffers, spin desalting columns and accessories
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.

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

References:
 

Drewett, J. G., et al. (1995). J. Biol. Chem. 270: 4668-4674.

Schmalzing, D., et al. (1995). Anal. Chem. 67(3):606.

Wolf, A., et al. (1994). J. Biol. Chem. 269: 23051-23058.

Milenic, D.E., et al. (1989). J. Immunol. Meth. 120: 71-83.

Rousseaux, J., et al. (1983). J. Immunol. Meth. 64: 141-146.

Coulter, A. and Harris, R. (1983). J. Immunol. Meth. 59: 199-203.

Mage, M.G. (1980). Meth. Enzymol. 70: 142-150.

 
 
 
 
 
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