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An improved method for immobilizing IgG antibodies on protein A-agarose.

Journal of immunological methods (1990-03-09)
T H Sisson, C W Castor
ABSTRACT

This report describes a modification of a procedure developed by others for crosslinking IgG to protein A which itself is covalently linked to a gel support. Earlier immunoaffinity columns were described as having large antigen-binding capacities and stability under a variety of elution conditions. The present data show that columns constructed with earlier techniques were only partially stable to pH 3.0 buffers, and, as a result, bound less than 20% of the antigen predicted by theory. Modifying parameters of the dimethylpimelimidate crosslinking method led to immunoaffinity columns which did not leak immunoglobulin under low pH elution buffer conditions. The new immunoaffinity absorbants, because of the increased strength of the couple between the antibody and protein A, were capable of binding antigen at over 80% of their theoretical capacity.

MATERIALS
Product Number
Brand
Product Description

Millipore
Protein A-Sepharose® from Staphylococcus aureus, lyophilized powder
Sigma-Aldrich
Triethanolamine borate, 97%