Ultracentrifugal sample preparation

There are various methods for the determination of the size distribution of organic pigment particles, the most common are sedimentation techniques in ultracentrifuges and specialized disk centrifuges as well as electron microscopy. These methods require considerable experimental skill, since the results depend largely on sample preparation and especially on the quality of the dispersion. 

In this section, we indicate the general factors that an experimentalist should consider while setting up a cation-mediated equilibrium RNA folding experiment, provide a stepwise protocol for sample preparation and outline the data collection procedure using the absorption optics of the analytical ultracentrifuge. 

To analyze free amino acids in plasma or tissue homogenates, it is necessary to remove proteins and peptides present in solution. The most widely used deproteinization method is precipitation with 5-sulfosalicylic acid followed by centrifugation for separating the precipitate. In comparison to other precipitation agents such as trichloroacetic acid, perchloric acid, picrinic acid, or acetonitrile, the best results with respect to completeness of precipitation are obtained with 5-sulfosalicylic acid [39]. Other deproteinization methods comprise ultrafiltration and ultracentrifugation [40], which have only recently been considered as sample preparation methods for amino acid analysis. 

The presence of proteins in biological fluids constitutes an interference in many analyses and their removal is often the major part of sample preparation. Proteins are typically removed by precipitation with acetonitrile or sulfosalicylic acid. Ultrafiltration or ultracentrifugation can also be used. 

The relation of the peak area (P ) of the elution pattern to lipid concentration in a loaded sample was examined using the standard lipoprotein (LDL) sample prepared by ultracentrifugation for three lipids, and a linear relationship was obtained in the following concentration ranges 5 - 54 pg for TC (Fig. 15), 17 - 51 pg for TG and 3 - 85 pg for PL. 

Bar = 50 nm. Samples were prepared at the initial concentrations of 2 raM lipid, 0.22 mM SQDG and 8pg Chla/ml PS II and concentrated by ultracentrifugation in the presence of Mes-Hepes/betaine buffer (pH 7.0). 

The usefulness of the ultracentrifuge in the preparation of samples rather than in the production of analytic data should not be overlooked. Preparative ultracentrifuges have utility in fractionating polymer samples and in freeing them from easily sedimented impurities. 

Spin the tubes at 12,000 rpm for 10 min in an SS34 rotor and save the supernatant. At this point if multiple tubes for the same organism or sample have been prepared, the supernatants can be combined. Do not exceed the volume of the ultracentrifuge tubes to be used for the cesium chloride gradient, which is usually 8 ml. When all supernatants have been combined, if the volume is not 8 ml then adjust to 8 ml by adding TE. 

---