328

23

Regulatory Networks

massive parallelization, but because of the current expense of transducing devices,

this parallelization is only practicable with protein microarrays, the penalty of which

is that almost all kinetic information is lost. Hence, at present, protein microarrays

and serial direct affinity measurement using biosensing devices are complementary

to each other. Miniaturization of the transducers and large-scale integration of arrays

of devices (comparable to the development of integrated circuit technology from

individual transistors, or the development of displays in which each pixel is driven

by a tiny circuit behind it), as in the Corning Epic system, allows the essential detailed

kinetic characterization to be carried out in a massively parallel mode. Significant

improvements in microarrays, allowing reliable kinetic information to be obtained

from them, are also envisaged. In effect, the two approaches will converge.

23.8.1

Chromatography

Chromatography denotes an arrangement whereby one binding partner is immobi-

lized to a solid support (the stationary phase) and the other partner is dissolved or

dispersed is a liquid flowing past the solid (the mobile phase). In essence, it is like the

biosensor; the difference is that binding is not measured in situ, but by depletion of

the concentration of the mobile partner in the output stream. As with the biosensor,

a drawback is that the immobilized protein has to be chemically modified in order to

be bound to the immobile phase of the separation system. In contrast to the biosen-

sor, the hydrodynamics within the column are complicated and chromatography is

not very useful for investigating the kinetics of binding. On the other, hand there is

usually an immense area of surface within the column, and the technique is therefore

useful for preparative purposes.

Typically, the protein complexes are identified using mass spectrometry (examples

of methods are tandem affinity purification, TAP, or high-throughput mass spectro-

metric protein complex identification, HMS-PCI; see Sect. 18.3).

23.8.2

Direct Affinity Measurement

As indicated in the legend to Fig. 23.1, a variety of transducers exist, the most popular

being the quartz crystal microbalance (QCM), surface plasmon resonance (SPR),

and optical waveguide lightmode spectroscopy (OWLS). ³⁰ A new and even more

sensitive technique is grating-coupled interferometry (GCI). ³¹ A great advantage

of biosensors is that no labelling of the interacting proteins is required, since the

transducers are highly sensitive. The order of intrinsic sensitivity is QCM less than< SPR

less than< OWLS less than< GCI. The most sensitive method until recently (i.e., OWLS) can easily

30 See Ramsden (1994) for a comprehensive survey of all these techniques and others.

31 Kozma et al. (2009).