Section 19.3.3.3.1. Sample preparation

Sample volumes required for one measurement are between 10 to 50 µl, occasionally more, depending on the specific design of the sample cell used. The concentration required to record a scattering curve with satisfactory statistics depends primarily on the molecular weight of the protein and the beam flux. Approximately 1 mg ml⁻¹ of a small protein (10–20 kDa) is the lower limit for recording a scattering pattern with satisfactory statistics when experiments are performed with a typical synchrotron instrument equipped with a gas-chamber detector. Somewhat lower concentrations of larger molecular weight proteins may be used. Higher concentrations will improve statistics significantly and reduce exposure times, but interparticle interference may result from high concentrations. Time-resolved experiments benefit dramatically from higher sample concentrations. In addition to the scattering power of the sample, the signal-to-background ratio and overall stability of an instrument (from X-ray source and optics to detector) limit the lowest concentration for a given experiment. Although higher concentrations add dramatically to the scattering and improve statistics, sample solutions must be monodisperse. Small-angle solution scattering is not well suited to the study of polydisperse systems, which give scattering of the entire molecular population weighted by the square of the mass, although a few distinct populations of substantially different sizes may be resolved with good-quality data. Chemical components that may have been carried along in a sample preparation, such as ammonium sulfate, sucrose, chloroform or caesium chloride, should be removed. The presence of such compounds may change the electron-density contrast and X-ray absorbency of the sample. In general, this can be most effectively avoided by exhaustive dialysis with the desired buffer solutions. The outer solution used for the final dialysis should be used for the blank measurements. Scattering contributions from the buffer solution, the sample cell and parasitic scattering must be subtracted from the measured scattering curve; these can be measured accurately from a well prepared blank. Extra buffer solution should be available for sample dilution. The data quality is improved and problems with radiation-sensitive samples are readily detected when protein concentrations and biological activities of samples are measured before and after the scattering experiment. Accurate protein concentration measurements permit scattering intensities from different samples to be scaled together accurately. This is particularly important in determining molecular weight.