Sapphire capture system

First-dimension analytical gel electrophoresis was performed, followed by second-dimension sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% fixed concentration gels, as previously described [47 (link),48 (link)]. The gels were scanned with a Sapphire Biomolecular Imager (Azure Biosystems, Dublin, OH, USA) and digitalized via the image analysis software Sapphire Capture system (Azure Biosystems, Dublin, OH, USA). Spot volumes were log-transformed to generate normally distributed data, and log-normalized volume instead of spot intensities was used in statistical processing to quantify differential expression. All spots were pre-filtered and manually checked before applying the statistical criteria (ANOVA test, p ≤ 0.05 and fold ≥ 1.5). Independent direct comparisons were made between the protein spots related to the OB, OD, and ODM groups, and the fold differences and p-values were calculated using one-way ANOVA. Spots that fulfilled the above-mentioned statistical criteria were submitted for further mass spectrometric (MS) analysis.

For the first dimension of separation, nine dry strips of Immobiline strips (measuring 24 cm and at a pH of 3–11; GE Healthcare, Danderyd, Sweden) were passively rehydrated (30 V; 12 h), followed by isoelectric focusing using an Ettan IPGphor IEF unit (GE Healthcare, Danderyd, Sweden). The focusing was performed at 20 °C, with 50 µA per strip. After the first dimension, the strips were equilibrated and separated on 12.5% SDS-PAGE gels using an Ettan Dalt Six device (GE Healthcare, Danderyd, Sweden). The gels were then scanned using a Sapphire Biomolecular Imager (Azure Bio systems, Dublin, OH, USA), and digitalization was performed using the image analysis software Sapphire Capture system (Azure Biosystems, Dublin, OH, USA). Preparative gels were prepared using the total protein (1 mg) obtained from a pool of equal protein amounts. The gels were then stained using Colloidal Coomassie Blue, as described previously [17 (link),30 (link),31 (link)].