Protein analyses and reagents: microscale assay of calcium-binding activity of proteins and peptides using a nitrocellulose membrane

Abstract

A simple and reliable method for the measurement of calcium binding to proteins and peptides was developed. It is composed of two procedures--filtration through a nitrocellulose membrane filter and estimation of 45Ca retained on the membrane. The routine assay was completed within a few minutes, and only microgram amounts of samples were necessary. This method permitted the quantitative determination of the calcium-binding activity of proteins and peptides including one with a molecular weight of as low as 4000. This method also permitted the detection of low-affinity interactions (Kd congruent to 10(-3) M), possibly because the nitrocellulose membrane did not show nonspecific binding of calcium and because a washing step was not employed in the routine assay. Ultrafiltration membranes when used in the apparatus gave no useful data.