*2.12. Protein Profiling by SDS-PAGE*

Ten samples of goldenrod honey and four reference honey samples (heather, honeydew, rape and multifloral) were prepared as in previous work with minor modifications [16]. One gram of raw honey was dissolved in 1 mL of deionized water containing 2% Nonidet P-40 substitute and 2% dithiothreitol. The samples were then mixed 2 to 1 with 4x concentrated standard Laemmli buffer and were incubated for 5 min at 95 ◦C. After cooling, 15 μL of the samples were applied to 15% denaturing gels (with 3% stacking gels). Electrophoresis was carried out on a Mini-Protean II apparatus (Bio-Rad Laboratories, Hercules, CA, USA) at 50 V for 15 min and then at 200 V for 1.5 h according to the standard method of Laemmli, with the BlueEasy Prestained Protein Marker (NIPPON Genetics EUROPE, Düren, Germany) as a molecular weight marker and Tris-Glycine-SDS buffer. After electrophoresis, gels were stained with Coomassie Brilliant Blue G-250. The staining was performed overnight and then gels were destained for 24 h with deionized water. Gels were scanned with Image Scanner III (GE Healthcare, Little Chalfont, UK) and processed by LabScan 6.0 (GE Healthcare, Little Chalfont, UK). Gels analysis was performed in ImageJ (1.52a) software to generate a graphical representation of each lane on the gel to assist in sample comparison. For better visualization, individual bands from the SDS-PAGE gel were contrasted with the profile obtained with the software.
