Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins.
The yield of antibody purified using affinity chromatography on immobilized Protein A or Protein G was increased up to 5-fold (500%) by including kosmotropic salts in the binding buffer. The binding buffer is used to equilibrate the affinity column before applying a sample to the column and also to dilute the sample prior to loading onto the affinity column to optimize conditions for a maximal binding of antibodies to affinity gels. In this study, the kosmotropic salts that were effective in greatly increasing antibody binding to Protein A included both inorganic and organic salts of ammonium; sodium; or potassium sulfate, phosphate, polycarboxylates; for example, succinate, citrate, isocitrate, N-(2-hydroxyethylene diamine triacetate (HEDTA), ethylene diamine tetraacetate (EDTA), and ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N'N'-tetra acetate(EGTA). On an equal-molar basis, the greater the number of carboxylic groups within the polycarboxylate molecule, the greater the increase in the yield of the purified antibody that was observed. The data show that kosmotropes can be used as effective additives to enhance the binding of immunoglobulins to Protein A or Protein G gels with a resultant increase in the yield of the purified antibodies. Thus, it appears that strongly hydrated anions (citrate, sulfate, and phosphate) and weakly hydrated cations (ammonium, potassium) increase the yield of antibody purified on either Protein A or Protein G affinity gels.
Ngo
That T
TT
BioProbe International, Inc., Tustin, CA, USA. thatngo@nttbiochem.com
Narinesingh
Dyer
D
D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods*; D005782:Gels / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification*; D051379:Mice; D010710:Phosphates / Q000737:chemistry; D012492:Salts / Q000737:chemistry*; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000276:immunology; D013431:Sulfates / Q000737:chemistry
D000906:Antibodies; D001426:Bacterial Proteins; D005782:Gels; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D010710:Phosphates; D012492:Salts; D013205:Staphylococcal Protein A; D013431:Sulfates; C024788:ammonium phosphate; C012036:sodium sulfate; D000645:Ammonium Sulfate