Linear gradient ipg strips

Total latex proteins were extracted as described (Wang et al., 2010 (link)). Protein concentration was determined by Bradford assay with BSA as a standard. For 2-DE, 1,300 μg proteins were loaded onto 24-cm, pH 3-10 linear gradient IPG strips (GE Healthcare, Uppsala, Sweden), and isoelectric focusing and gel electrophoresis were performed as described (Wang et al., 2013 (link)). Three biological replicates were performed for each sample. Protein spots in gel were visualized by the CBB-G250 staining, and images were analyzed with the ImageMaster 2D Platinum software (GE Healthcare). The average Vol% for each matched protein spot was calculated based on all the detected gel images from the three biological repeats (Supplementary Figure S1). First, we compared Eth, Mo, and EMo with CK, as well as EMo with Eth and EMo with Mo; the protein spots that, in at least one pair, showed an at least 1.5-fold change in abundance (p < 0.05) were termed as differentially expressed protein (DEP) spots. Then, these protein spots were positively identified by MALDI TOF MS/MS.

Latex proteins were extracted as described33 (link). Protein concentration was determined by Bradford assay with BSA as a standard. For common 2-DE, 1,000 μg of proteins were loaded onto the 24-cm, pH 4-7 linear gradient IPG strips (GE Healthcare, Uppsala, Sweden), and then, isoelectric focusing and gel electrophoresised were performed as described19 64 (link). Three biological replicates for each separation were conducted.
2-D DIGE analysis was performed as described64 (link). Proteins from the D-3, E-3, D-5 and E-5 latex samples were labeled with a ratio of 400 pmol Cy3 or Cy5 per 50 μg of proteins. For gel normalization, an internal standard was prepared by pooling an equal protein quantity from each of the four samples and labeling with Cy2. Then, the Cy2-, Cy3- and Cy5-labeled 2-DE images were acquired using a Typhoon Trio scanner (GE Healthcare, Piscataway, NJ, USA), and the DIGE images were analyzed using DeCyder 7.0 software (GE Healthcare). A differential in-gel analysis module was used for spot detection, and a biological variation analysis module was applied to the three biological repeats to identify the differentially expressed spots with higher than 95% confidence.