This study was designed to identify capsule components by full-scan mass spectrometry analyses coupled to liquid chromatography based on a single injection of the capsule material previously solubilized in methanol.
Following this washing step the material was solubilized in 500 µl of urea lysis buffer (7.5 M Urea, 2.5 M Thiourea, 40 mM Tris pH 7.6, 2.5% Octyl-b-glucoside, 6.25 mM TCEP, 1.25× Proteinase inhibitor) followed by homogenization on ice 10 times using a Potter homogenizer.
Insoluble material was solubilized by vigorous agitation in 6 M GuHCl, re-homogenized and left for four hours at room temperature (RT) before storage at −80°C.
After sonication, DDM was added to 0.5%, and lysed material was solubilized for 1 h on a rotary platform in a cold room (4 °C).
Insoluble material was solubilized by vigorous agitation in 6 M GuHCl, re-homogenized and left for 4 h at room temperature (RT) before storage at −80°C.Total protein concentrations were determined using the Total Protein Kit (Sigma Aldrich, Dorset, UK) following the manufacturer's guidelines.
TBS-soluble materials were solubilized in Laemmli sample buffer and subjected to SDS-PAGE.
To create insoluble extracts, RIPA-insoluble pelleted material was first solubilized in 100 μl 50 mM Hepes, pH 7.0, 150 mM NaCl, 1% SDS and 10% DMSO, then diluted with 0.9 ml of SDS-free RIPA, spun for 10 min, and supernatants were transferred to clean tubes.
The products of microbial decomposition of organic materials might solubilize some of the sorbed or recalcitrant P in soil causing an increase in organic P associated with the humic substances (Reddy et al. 2005).
After washing away the unbound biotinylation reagent, membranes were prepared and solubilized and this material was subjected to streptavidin precipitation and SDS/PAGE resolution of the precipitated proteins.
The resulting lysates were clarified by centrifugation (3200 x g), and after removal of the soluble material the insoluble pellet was solubilized with 4 M urea.
