Protein A or Protein G 993

142
Purification of IgG Using Protein A or Protein G

Mark Page and Robin Thorpe

1. Introduction
Some strains of Staphylococcus aureus synthesize protein A, a group-specific ligand
that binds to the Fc region of IgG from many species (1,2). Protein A does not bind all
subclasses of IgG, e.g., human IgG3, mouse IgG3, sheep IgG1, and some subclasses
bind only weakly, e.g., mouse IgG1. For some species, IgG does not bind to protein A
at all, e.g., rat, chicken, goat, and some MAbs show abnormal affinity for the protein.
These properties make the use of protein A for IgG purification limited in certain
cases, although it can be used to an advantage in separating IgG subclasses from mouse
serum (3). Protein G (derived from groups C and G Streptococci) also binds to IgG Fc
with some differences in species specificity from protein A. Protein G binds to IgG of
most species, including rat and goat, and recognizes most subclasses (including human
IgG3 and mouse IgG1), but has a lower binding capacity. Protein G also has a high affinity
for albumin, although recombinant DNA forms now exist in which the albumin-binding
site has been spliced out, and are therefore very useful for affinity chromatography. Other
streptococcal immunoglobulin binding proteins are protein H (binds IgG Fc), protein B,
which binds IgA and protein Arp, which binds IgG & IgA. These are not generally
available for immunochemical use.
Another bacterial IgG-binding protein (protein L) has been identified (4). Derived
from Peptostreptococcus magnus, it binds to some κ (but not λ) chains. Furthermore,
protein L binds to only some light-chain subtypes, although immunoglobulins from
many species are recognized (5,6).
Finally, hybrid molecules produced by recombinant DNA procedures, comprising
the appropriate regions of IgG-binding proteins (e.g., protein L/G, protein L/A) also
have considerable scope in immunochemical techniques. These proteins are therefore
very useful in the purification of IgG by affinity chromatography. Columns are com-
mercially available (MabTrap G II, Pharmacia, Uppsala, Sweden) or can be prepared
in the laboratory. The product of this method is of high purity and is useful for most
immunochemical procedures including affinity chromatography and conjugation with
radioisotopes, enzymes, biotin, and so forth.

From: The Protein Protocols Handbook, 2nd Edition

Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ

993
994 Page and Thorpe

2. Materials
1. PBS: 0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH2 PO4, 8.1 mM Na2HPO4.
2. Sodium azide.
3. Dissociating buffer: 0.1 M glycine-HCl, pH 3.5. Adjust the pH with 2 M HCl.
4. 1 M Tris.
5. Binding buffer: Optimal binding performance occurs using a buffer system between pH 7.5
and 8.0. Suggested buffers include 0.1 M Tris-HCl, 0.15 M NaCl, pH 7.5; 0.05 M sodium
borate, 0.15 M NaCl, pH 8.0; and 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5.
6. IgG preparation: serum, ascitic fluid, or hybridoma culture supernatant.
7. Protein A, G column.

3. Methods
Refer to Chapter 144 for CNBr activation of Sepharose and coupling of protein A, G.
1. Wash the column with an appropriate binding buffer.
2. Pre-elute the column with dissociating buffer, 0.1 M glycine-HCl, pH 3.5.
3. Equilibrate the column with binding buffer.
4. Prepare IgG sample: If the preparation is serum, plasma, or ascitic fluid, dilute it at least
1:1 in binding buffer and filter through 0.45-μm filter. Salt-fractionated preparations
(see Chapter 137) do not require dilution, but the protein concentration should be adjusted
to approx 1–5 mg/mL. Hybridoma culture supernatants do not require dilution.
5. Apply sample to column at no more than 10 mg IgG/2-mL column.
6. Wash the column with binding buffer until the absorbance at 280 nm is <0.02.
7. Dissociate the IgG-ligand interaction by eluting with dissociating buffer. Monitor the
absorbance at 280 nm, and collect the protein peak. Neutralize immediately with alkali
(e.g., 1 M Tris, unbuffered).
8. Wash the column with binding buffer until the pH returns to that of the binding buffer.
Store the column in buffer containing at least 0.15 M NaCl and 0.1% sodium azide.
9. Dialyze the IgG preparation against a suitable buffer (e.g., PBS) to remove glycine/Tris.

References
1. Lindmark, R., Thorén-Tolling, K., and Sjöquist, J. (1983) Binding of immunoglobulins to
protein A and immunoglobulin levels in mammalian sera. J. Immunol. Meth. 62, 1–13.
2. Hermanson, G. T., Mallia, A. K., and Smith, P. K. (1992) Immobilized Affinity Ligand
Techniques. Academic, San Diego, CA.
3. Ey, P. L., Prowse, S. J., and Jenkin, C. R. (1978) Isolation of pure IgG1, IgG2a, and IgG2b
immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry 15,
429–436.
4. Kerr, M. A., Loomes, L. M., and Thorpe, S. J. (1994) Purification and fragmentation of
immunoglobulins, in Immunochemistry Labfax (Kerr, M. A. and Thorpe, R., eds.), Bios
Scientific, Oxford, UK, pp. 83–114.
5. De Chateau, M., Nilson, B. H., Erntell, M., Myhre, E., Magnusson, C. G., Akerstrom, B.,
and Bjorck, L. (1993) On the interaction between protein L and immunoglobulins of
various mammalian species. Scand. J. Immunol. 37, 399–405.
6. Akerstrom, B., Nilson, B. H., Hoogenboom, H. R., and Bjorck, L. (1994) On the interac-
tion between single chain Fv antibodies and bacterial immunoglobulin-binding proteins.
J. Immunol. Meth. 177, 151–163.