Antibody capture with Reacti-Bind Coated Protein A, G, A/G and L Plates

Highlights of using Reacti-Bind Plates for antibody capture:

• Retain antibody activity, which can be lost when antibodies are immobilized by passive adsorption
• Orient antibodies for maximum antigen-binding capacity
• Immobilize antibodies without prior purification
• Ensure minimal variation (<5% well-to-well) from consistent coating
• Reduce nonspecific binding because plates are pre-blocked with SuperBlock Blocking Buffer
• Perform multiple immunoprecipitations
• Binds up to 2.5 µg antibody/well (100 µl coating volume)

Protein A/G

• Consists of a fusion protein containing four antibody-binding sites from Protein A and two from Protein G
• Binds to the broadest spectrum of antibodies because it combines the specificities of Protein A and Protein G
• Binds to Fc region of antibodies, ensuring optimal orientation
• Binds well using a wide range of pH

Protein A

• Binds strongly to IgG from rabbit, guinea pig, pig, dog and rhesus monkey
• Binds strongly to mouse IgG2a, IgG2b and IgG3
• Binds to Fc region of antibodies for optimal orientation

Protein G

• Binds strongly to IgG from many species including human, mouse, rabbit, sheep and goat
• Binds only to IgG – no cross-reactivity with other antibody classes
• Binds to Fc region of antibodies for optimal orientation

• Binds to all classes of immunoglobulins (IgG, IgM, IgA, IgE and IgD)
• Binds to the VL region of kappa light chains (human I, III, IV and Mouse I) without interfering with antigen-binding sites
• Binds to Fab fragments and ScFv
• Does not bind bovine, goat or sheep immunoglobulin
• Binds weakly to rabbit immunoglobulin
• Pre-blocked to reduce nonspecific binding

Figure 1. Comparison of signal obtained by antibody bound to Protein A and Protein G coated plates, and antibody passively bound to polystyrene plates. Goat Anti-Biotin was serially diluted across Protein A, Protein G and polystyrene plates. The Goat Anti-Biotin was detected by the addition of biotin, followed by streptavidin-HRP and finally 1-Step Turbo TMB. 

Comparison of Protein A, G, A/G and L Immunoglobulin Binding Proteins

Protein A References:

1. Lawrenson, I.D., et al. (2002). J. Cell Sci. 115, 1059-1072.
2. Asthagiri, A.R., et al. (1999). J. Biol. Chem. 274, 27119-27127.

Protein G References:

1. Rauch, J., et al. (2003). J. Biol. Chem. 278, 47508-47515.
2. Lai, Z., et al. (2002). Proc. Natl. Acad. Sci. USA 99, 14734-14739.

Protein L References:

1. Björck, L. (1988). J. Immunol. 140, 1194-1197.
2. Kastern, W., et al. (1992). J. Biol. Chem. 267, 12820-12825.
3. Nilson, B.H.K., et al. (1993). J. Immunol. Methods 164, 33-40.
4. Åkerström, B. and Björck, L. (1989). J. Biol. Chem. 264, 19740-19746.
5. Rhee, J., et al. (2001). J. Biol. Chem. 276, 6640-6644.