Protein microarray technology
This approach to the study of receptor–ligand binding exploits the principle that
assay systems that use a small amount of capture molecules (the ligand) and a small
amount of target molecules (the receptor) can be more sensitive than systems that
use a hundred times more material (Section 8.5.5). In this miniaturisation approach,
the ligand is immobilised onto a small area of a solid phase, commonly a derivatised
glass slide. The resulting ‘microspot’ contains a high density (concentration) of ligand
but a very small amount of it. It is then incubated with the receptor, commonly
fluorescently tagged, resulting in the binding of some of the receptor molecules.
Since the microspot covers a small area there is effectively no change in the
concentration of the unbound ligand in the sample even if its concentration was
low and the binding affinity was high. This is true provided that<0.1Kdof the ligand
molecules are bound in the complex, whereKdis the dissociation constant for the
complex. The ligand–receptor complex is then quantified by fluorimetric methods
and the procedure repeated for a series of increasing microspot sizes (increasing
ligand concentration) but such that the density of the capture molecules is constant.
After each incubation excess receptor protein is washed away and the remaining
complex analysed by surface-enhanced laser desorption/ionisation (SELDI) mass
spectrometry, a variant of MALDI (Section 9.3.8). In practice, microspots are immo-
bilised in rows on the solid support allowing the simultaneous analysis of hundreds
or even thousands of samples.Other methods used to study ligand–receptor binding
Other experimental methods that can be used to quantify receptor–ligand binding and
hence characterise both the receptor and the ligand include:- analytical ultracentrifugation by the sedimentation velocity (Section 3.5.1),
- scintillation proximity assay (Section 14.3.2) commonly using^3 H- or^125 I-labelled
ligand, - NMR (Section 13.5) observing either changes in the signal of the protein or of the
ligand induced by receptor–ligand binding, - X-ray crystallography (Section 13.6) either by co-crystallising the receptor and ligand
or by soaking crystals of the receptor in solutions of the ligand.
17.4 Mechanisms of signal transduction
17.4.1 Cell signalling assays
In order to determine the details of a specific transduction pathway it is necessary to
identify the proteins and other small effectors whose activity or concentration
change in response to the activation of the receptor. This is possible usingfunc-
tional screening assaysthat are of two main types:cell-free(biochemical) andcell-
based(cellular) (see Section 18.2.3 for further details). In practice, whilst cell-free685 17.4 Mechanisms of signal transduction