Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

104 Julenius
Fig. 1. Under conditions of total internal reflection at a metal-coated interface, an
evanescent wave propagates into the medium of lower refractive index. Surface plasmon
resonance is observed as a sharp dip in reflected intensity at an angle which depends
on the refractive index of the medium on the nonilluminated side of the surface.
length of the incident light, and the refractive index of the media on either side
of the metal film. If the metal film properties, wavelength, and refractive index
of the denser medium is kept constant, the SPR signal can be used to probe the
refractive index of the medium into which the evanescent wave propagates (the
nonilluminated side of the surface), as is done in the commercial applications.
The SPR instrument utilizes a sensor chip, which consists of a glass slide
with a thin layer of metal on one side. The metal film is covered with a matrix,
to which the macromolecules are immobilized. The chip can be replaced, making
it possible to use different kind of matrices depending on the nature of the
molecule to be immobilized (see Note 1). The need for substantial changes in
refractive index upon binding limits the use to macromolecules; i.e., it is not
possible to measure calcium-binding directly. But it is possible to study the
calcium dependence of interactions between macromolecules by performing
experiments in the absence and presence of calcium. This chapter will describe
the testing of calcium-dependence of a protein–protein association reaction
using standard coupling methods on a carboxymethylated dextran matrix chip.
It will also describe a quantitative kinetic experiment.
2. Materials
1. SPR instrument. See Note 2 for information about commercial instruments.
2. Sensor chip with carboxymethylated dextran matrix surface. These are bought
from the manufacturer of the SPR instrument. BIAcore calls this chip CM5.
3. Normalization solution: glycerol dissolved in distilled water is needed if the sensor
chip is new. The glycerol concentration needed is instrument-dependent, see
your instrument manual.