Sample preparation is a fundamental step in proteomic methodologies. The quality of the results from a proteomic experiment is dependent on the nature of the sample and the properties of the proteins. In this study, various pre-treatment methods were compared by proteomic analysis; we analysed various rat brain structures after chloroform/methanol, acetone, TCA/acetone and TCA protein precipitation procedures. The protein content of the supernatant was also examined by 2-DE. We found that for four of the rat brain structures, precipitation with chloroform/methanol and acetone delivered the highest protein recovery for top-down proteomic analysis; however, TCA precipitation resulted in good protein separation and the highest number of protein spots in 2-DE. Moreover, TCA precipitation also gave high efficiency of protein recovery if prior sonication procedure was performed.

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Lactoglobulin is a natural protein present in bovine milk and common component of human diet, known for binding with high affinity wide range of hydrophobic compounds, among them fatty acids 12-20 carbon atoms long. Shorter fatty acids were reported as not binding to β-lactoglobulin. We used X-ray crystallography and fluorescence spectroscopy to show that lactoglobulin binds also 8- and 10-carbon caprylic and capric acids, however with lower affinity. The determined apparent association constant for lactoglobulin complex with caprylic acid is 10.8 ± 1.7 × 10(3) M(-1), while for capric acid is 6.0 ± 0.5 × 10(3) M(-1). In crystal structures determined with resolution 1.9 Å the caprylic acid is bound in upper part of central calyx near polar residues located at CD loop, while the capric acid is buried deeper in the calyx bottom and does not interact with polar residues at CD loop. In both structures, water molecule hydrogen-bonded to carboxyl group of fatty acid is observed. Different location of ligands in the binding site indicates that competition between polar and hydrophobic interactions is an important factor determining position of the ligand in β-barrel.