Section 19.4.5. Practical considerations

For a biological scientist, the first question is whether knowledge of the parameters that can be obtained from neutron scattering is of interest for the biological problem under consideration. If the answer is affirmative, the best course of action is to make contact with individuals who have conducted experiments in the past, as well as with biologists at a neutron-scattering facility. As a preliminary guide, a few general points are pertinent.

For a solution-scattering experiment on an unlabelled sample, typical sample volumes are 0.1–0.2 ml at concentrations of around 10 mg ml⁻¹. For contrast variation, samples dialysed to different D₂O levels will be required, so significant amounts of material need to be at hand. Making a set of measurements will require a few hours at a modern facility.

Longer collection times and significantly greater biochemical efforts will be involved for measurements with labelled material, especially if reconstitution is part of the strategy. Labelled biomolecules are most often produced by growth of organisms in D₂O (Moore & Engelman, 1976; Vanatalu et al., 1993), but strategies using chemical synthesis or providing labelled precursors have also been employed. Preparation of samples with labelled ligands is usually more straightforward than the generation of reconstituted complexes, but still requires tests of homogeneity.

For measurements to be made, the samples must be transported to a neutron facility and measured for many hours. This requires stability in the usual biochemical sense, and proper experiments will include assays performed after the measurements. Damage from the neutrons themselves is minimal, but handling and transportation impose greater challenges.

Where mixed samples are employed, stability also involves resistance to exchange of subunits or ligands between complexes in the mixture. This is hard to assess in advance, the first sign of trouble often being an absence of an expected difference signal. Given shipment and measuring times, even a slow exchange is significant. With a consequent qualification of the results, cross-linking may be used to stabilize the complex.

In all scattering measurements, homogeneity of the sample is a great advantage in interpretation. However, some variation is tolerable for many purposes, and the level of purity typically sought for crystallization experiments is not usually required.

Background arises from the incoherent scattering of hydrogen. Consequently, the use of high levels of D₂O in the buffer improves the background, but may not be innocuous, since high levels of D₂O have been seen to induce aggregation. In cases where complexes with labelled subunits are measured, it is advantageous to suppress small-angle background scatter by contrast matching the unlabelled regions via solvent adjustment.