《Protein characterization by static light scattering》.pdf

中国试剂网 2 . 4 . 2 1 Protein characterization by static light scattering Introduction to static light scattering Static light scattering (SLS) is a non-invasive technique whereby an absolute molecular mass of a protein sample in solution may be experimentally determined to an accuracy of better than 5% through exposure to low intensity laser light (690 nm). The intensity of the scattered light is measured as a function of angle and may be analyzed to yield the molar mass, root mean square radius, and second virial coefficient (A2). The results of an SLS experiments can be used as a quality control in protein preparation (e.g. for structural studies) in addition to the determination of solution oligomeric state (monomer/dimer etc.). SLS experiments may be performed in either batch or chromatography modes. However, as the measurement yields the volume-averaged molecular weight of the sample within the laser beam it is more powerful to utilize the technique in combination with protein purification. As the measurements are performed in a flow cell there is no loss of sample and the SLS detector can easily be integrated into standard protein purification equipment. Due to the necessity of obtaining good baselines in both 280 nm absorption measurements (UV) and light scattering (LS) measurements, SEC represents a good choice of separation media, due to the use of only a single buffer system for the entire purification. Since the light scattering and concentration are measured for each eluting fraction, the mass and size can be determined independently of the elution position. This is particularly important for protein species with non-globular shapes, which may elute at positions distant from that predicted by the calibration curve for the column. Materials 1.