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.
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.
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 also 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 – 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
Product Details:
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.
Digestion and purification scheme for preparing Fab fragments from IgG antibodies.
Typical SDS-PAGE (10% Bis-Tris) result of reduced and non-reduced Fab and Fc fragments from rabbit IgG.
Recommended digestion times for various species and concentrations of IgG.
Digestion Time (hours)
[IgG] (mg/mL)
Rabbit IgG
Human IgG
Mouse IgG
8
8 to 9
5 to 6
4 to 5
4
6 to 7
5 to 6
3 to 4
1.5
4 to 5
3 to 4
2 to 3
0.5
3 to 4
2 to 3
2 to 3
References:
Lindner I. et al. (2010) {alpha}2-Macroglobulin inhibits the malignant properties of astrocytoma cells by impeding {beta}-catenin signaling. Cancer Res. 70, 277-87.
Kaufmann B. et al. (2010) Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354. PNAS. 107, 18950-5.
Chen X. et al. (2010) Requirement of open headpiece conformation for activation of leukocyte integrin {alpha}x{beta}2. PNAS. 107, 14727-32.
Uysal H. et al. (2009) Structure and pathogenicity of antibodies specific for citrullinated collagen type II in experimental arthitis. J. Exp. Med. 206, 449-62.
Thomas G. M. et al. (2009) Cancer cell-derived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo. J. Exp. Med. 206, 1913-27.
Kong F. et al. (2009) Demonstration of catch bonds between an integrin and its ligand. J. Cell Biol. 185, 1275-84.
