Sequential volume changes on a small sample by density

A procedure is described for generating a sequence of volume changes by density on the same sample of a solution. The entire volume-change profile over the reactant concentration range of interest can be accomplished with 0.1-0.2 mumol of a protein in 100 microliters of sample. The calcium-binding proteins, calmodulin and skeletal troponin-C, were employed to determine the volume increases attending the sequential addition of calcium ions. Both volume-change profiles exhibited nonlinear increases over the first four equivalents of calcium ion added.