Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

4 Yazawa
Fig. 1. Schematic representation of a flow-dialysis cell.
ing a protein solution is separated by the cellulose membrane from the buffer
chamber filled with the buffer solution (see Fig. 1). Each solution in both of the
chambers is continuously mixed with a magnetic stirring bar, and the concentration
of free ligand in the protein solution is determined based on the rate of
diffusion into the buffer chamber, which is proportional to the concentration of
free ligand. The buffer chamber is connected to the reservoir and is flushed
continuously with the fresh buffer solution at a constant rate and the outlet is
connected to a fraction collector to monitor the radioactivity in the effluent.
When small amounts of the labeled ligand in a small volume are added to the
sample chamber, chemical equilibrium is attained usually within a fraction of a
second and the ligand molecules free from the protein molecule diffuse into the
buffer chamber at a rate depending on the equilibrium concentration and the
characteristics of the membrane. Under the constant flow rate of the buffer
solution in the buffer chamber, the radioactivity in the buffer chamber becomes
constant in a matter of minutes when the steady state is reached, which can be
a measure of the concentration of the free ligand in the sample chamber. Then
small amounts of the unlabeled ligand in a small volume are added to the
sample chamber, a new chemical equilibrium is attained together with a rapid
exchange between isotopes, and the free ligand diffuses at a different rate
depending on the concentration in the sample chamber giving a new steadystate
level of radioactivity in the effluent. After successive additions of the
unlabeled ligand, followed by determinations of the respective steady-state levels
of the radioactivity, excess unlabeled ligand in a small volume is added, a
maximum value for the radioactivity (C n) in the effluent is reached. This can
correspond to that expected when no appreciable fraction of the labeled ligand
is bound. That is, this value becomes a measure of the total concentration of the