23.8 In Vitro Experimental Methods

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undesirable features, such as the need to label the proteins and the possibility of

unfavourable alignment of the fluorophores, such that energy transfer is hindered

even though A and B are indeed associated.

RNA–protein binding can be investigated by the systematic evolution of ligands by

the exponential enrichment (SELEX) technique, in which candidate RNA oligomers

(possibly initially random) are passed through an affinity column of the protein of

interest. Retained RNA is eluted, amplified using PCR, and reapplied to the column.

The cycle is repeated until most of the RNA binds, whereupon it is sequenced.

23.8 In Vitro Experimental Methods

Here affinities are measured outside the cell. At least one of the proteins of interest

has to be isolated and purified. It can then be immobilized on a chromatographic

column and the entire cell contents passed through the column. Any other proteins

interacting with the target protein will be bound to the column and can be identified

after elution.

A much more powerful approach, because it allows precise characterization of

the kinetics of both association and dissociation, is to immobilize the purified target

protein on a transducer able to respond to the presence of proteins binding to the target.

The combination of capture layer and transducer is called a biosensor (Fig. 23.1).

Although this approach is formally in vitro, the physiological milieu can be repro-

duced to practically any level of detail. Indeed, as pointed out in the introduction to

this chapter, the microenvironment of a subcellular compartment can be more pre-

cisely investigated than in vivo. Nevertheless, since each interaction is individually

measured, with as much detail as is required, high throughput is only possible with

Fig. 23.1 Schematic representation of a biosensor. The thickness and structure of the capture

layer, which concentrates the analyte, whose presence can then be registered by the transducer,

largely determines the temporal response. The main transducer types are mechanical (cantilevers, the

quartz crystal microbalance), electrical (electrodes, field-effect transistors), optoelectronic (surface

plasmon resonance), and optical (planar waveguides, optical fibres). See Ramsden (1994) and

Scheller and Schubert (1989) for comprehensive overviews.