Optimization of binding capacity and specificity of protein G on various solid matrices for immunoglobulins.
Streptococcal protein G is a more versatile and efficient alternative to staphylococcal protein A in purifying immunoglobin G (IgG) isotypes from various animal species. Optimizations are most dramatic with goat IgG, which binds protein G 55 times better than protein A. Using GammaBind G (a recombinant form of protein G (Genex Corp.)), we optimized binding capacity and specificity for IgG. Protein G was covalently coupled to three different matrices (CNBr-Sepharose, Tresyl-Sepharose, and Affigel-10) and compared with protein A-CNBr-Sepharose. Equal volumes of human, mouse, and goat serum samples were equilibrated into Hepes/NaOH buffers with various ionic strengths (i.e., concentrations of NaCl) and pH values and allowed to bind to affinity columns of proteins G and A. Bound ligands were eluted with 8.0 M urea, 0.05-M Tris/HCl, pH 8.00. Bound fractions were assayed for protein concentration and analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis. The optimal conditions for binding IgG to protein G are 1.0 M NaCl and pH 8.0 for human, mouse, and goat.
Pilcher
J B
JB
Division of Parasitic Diseases, Centers for Disease Control, Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA 30333.
Tsang
V C
VC
Zhou
W
W
Black
C M
CM
Sidman
C
C
D000818:Animals; D000915:Antibody Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006041:Goats; D006801:Humans; D007070:Immunoglobulin A; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007132:Immunoglobulin Isotypes / Q000302:isolation & purification*; D066298:In Vitro Techniques; D051379:Mice; D009419:Nerve Tissue Proteins
C029152:G-substrate; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D009419:Nerve Tissue Proteins