2.3.2. Protein Profile

The two-dimensional (2D) gel analysis was carried out according to Kumar et al. (2017), with modifications [29]. Briefly, 2 mg of proteins from the four *T. molitor* fractions were mixed with the sample buffer containing 7 M urea, 2 M thiourea, 4% CHAPS (*w*/*v*), 1% Triton-X-100 (*v*/*v*), 20 mM Tris, 1% DTT (*w*/*v*) and 0.5% pH 3-10 IPG buffer (*v*/*v*). A volume of 250 µL of each sample solution (TMI, TMD, HDSP and NHSP) was loaded on a 13 cm GE Healthcare Immobiline® DryStrip (pH 3–10) and first-dimension isoelectric

focusing (IEF) was performed using the Ettan IPGphor 3 IEF system (GE Healthcare, Piscataway, NJ, USA) at 20 ◦C, 50 µA/strip, 30 V for 12 h (rehydration), 100 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 5000 V for 1 h and then 8000 V until 16,000 Vh was reached. The first-dimension strips were kept at −20 ◦C until used for the second dimension. For the second dimension, each strip was soaked in 20 mL of equilibration buffer containing 6 M urea, 2 M Milli-Q H2O, 50 mM Tris-HCl pH 8.8, 30% glycerol (*v*/*v*), 2% SDS (*v*/*v*), 2% DTT (*w*/*v*) and traces of bromophenol blue for 15 min at 20 ◦C. SDS-PAGE was carried out at 10 ◦C using 17.5 cm × 16.8 cm × 1 mm 15% polyacrylamide gels topped with 5% polyacrylamide stacking gels. The migration was performed at 25 µA for each gel. The running buffer consisted of 1X tris-glycine SDS solution (Bio-Rad, Hercules, CA, USA). The molecular weights (MW) of mealworm proteins were estimated by using a MW marker (Prestained Protein Standards Precision Plus Protein™ All Blue, Bio-Rad, cat. #1610373, Hercules, CA, USA). Proteins were fixed by soaking the gels in a 50% MeOH (*v*/*v*) and 10% acetic acid (*v*/*v*) solution for 12 h. Gels were then stained by soaking in GelCode™ Blue Stain Reagent (Thermo Fisher Scientific, Whaltham, MA, United-States) for 24 h, rinsed in Milli-Q water and scanned on a ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA). The 2-D gel image analysis was performed using ImageJ software according to the procedure of Natale et al. (2011), with modifications [30]. Briefly, gel images were aligned using the same image as reference with the bUnwarpJ plugin and repetitions for TMI, TMD, HDSP, and NDSP were stacked and summed to give a single gel image representative of all 3 repetitions.
