TY - JOUR
T1 - [21] Measurement of Ligand-Protein Interaction by Electrophoretic and Spectroscopic Techniques
AU - Oberfelder, Robert W.
AU - Lee, James C.
PY - 1985/1/1
Y1 - 1985/1/1
N2 - Determination of the equilibrium binding constant and the stoichiometry for a protein-ligand interaction requires an appropriate choice of methodology. Each technique has advantages and disadvantages, and practical and theoretical limitations, and these must be considered when choosing a procedure. The chapter illustrates that it is important to have a number of approaches available from which to choose, so that techniques can be used which is compatible with the constraints placed on the system because of the nature of the protein and ligand of interest. Several procedures have been discussed in this chapter including equilibrium dialysis. In these methods the formation of a ligand-protein complex is detected by directly determining the difference in ligand concentrations. Indirect approaches utilizing absorbance spectroscopy, fluorescence spectroscopy, or electrophoresis may also be employed to perform binding studies. These studies use changes in the spectral properties of the protein or ligand to measure equilibrium binding constants. The procedures used to perform binding studies utilizing spectroscopy, electrophoresis, and isoelectric focusing are addressed and the advantages and disadvantages intrinsic to these techniques is also described in the chapter.
AB - Determination of the equilibrium binding constant and the stoichiometry for a protein-ligand interaction requires an appropriate choice of methodology. Each technique has advantages and disadvantages, and practical and theoretical limitations, and these must be considered when choosing a procedure. The chapter illustrates that it is important to have a number of approaches available from which to choose, so that techniques can be used which is compatible with the constraints placed on the system because of the nature of the protein and ligand of interest. Several procedures have been discussed in this chapter including equilibrium dialysis. In these methods the formation of a ligand-protein complex is detected by directly determining the difference in ligand concentrations. Indirect approaches utilizing absorbance spectroscopy, fluorescence spectroscopy, or electrophoresis may also be employed to perform binding studies. These studies use changes in the spectral properties of the protein or ligand to measure equilibrium binding constants. The procedures used to perform binding studies utilizing spectroscopy, electrophoresis, and isoelectric focusing are addressed and the advantages and disadvantages intrinsic to these techniques is also described in the chapter.
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U2 - 10.1016/S0076-6879(85)17023-0
DO - 10.1016/S0076-6879(85)17023-0
M3 - Article
C2 - 4079810
AN - SCOPUS:0022330807
SN - 0076-6879
VL - 117
SP - 381
EP - 399
JO - Methods in enzymology
JF - Methods in enzymology
IS - C
ER -