Ganglia were partially thawed, the outer fascia removed by dissection, and a portion macerated with a scalpel before partial homogenization in either radioimmune precipitation assay buffer with protease inhibitor mixture (Roche) for QFWB (see below) or iTRAQ extraction buffer containing 6 m Urea, 2 m thiourea, 2% CHAPS, 0.5% SDS, and protease inhibitor mixture (Roche) for proteomic processing (see below). Samples were pooled by condition and manually homogenized in a dounce glass homogenizer. Homogenized samples were sonicated in a cup style sonicator six times for 15 s at power level 7.5 with vortexing for 30 s between each round of sonication. Samples were left on ice for 10 min before being revortexed then centrifuged at 20,000 g for 30 min at 4 °C. The resulting pellet containing proteins insoluble when processed in this manner was stored at −80 °C, and the supernatant was transferred to a fresh 1.5 ml tube to be processed for iTRAQ labeling as previously described (17 (link)20 (link, link, link)).
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