49] Preparation of contractile proteins for photon correlation spectroscopic and classical light-scattering studies

Abstract

Publisher Summary This chapter discusses the preparation of contractile proteins for photon correlation spectroscopic and classical light-scattering studies. The development of photon correlation spectroscopy (PCS) has made possible the precise measurements of translational and rotational diffusion coefficients of proteins, nucleic acids, and other particles of biological origin. It is also possible to conduct classical light-scattering measurements of the molecular weight, radius of gyration, and second virial coefficient with a photon correlation spectrometer. Because such spectrometers employ lasers, they provide a monochromatic, constant-intensity light source and a highly collimated beam that is ideally suited for light scattering on extremely dilute macromolecular solutions. The reliability of any light-scattering or PCS measurement is critically dependent upon the absence of large, unwanted, contaminants, henceforth referred to as dust, regardless of origin. To keep dust contamination at a minimum, a few general procedures must be employed. For aqueous solutions, distilled water should be deionized and filtered with the equivalent of a Millipore Milli-Q reagent grade water system equipped with a Twin-90 filter. This system removes dust particles greater than 0.22 μm in diameter. It is also found that precise PCS measurements of diffusion coefficients require both accurate determination of the scattering angle and temperature regulation to ±0.05 ° or better.