Powerpac 300

Each amplified DNA fragment generated by semi-quantitative PCR was separated via agarose gel (2%) (Cleaver Scientific, Warwickshire, UK) electrophoresis. Gel was prepared by diluting agarose in tris-borate-EDTA buffer (TBE) (45 mM TrisHCl, 0.45 M boric acid, 10 mM EDTA) (Merck, Ñuñoa, Chile) buffer with the pH adjusted to 8. To visualize DNA bands, 0.5 µL of GelRed (Biotium, Fremont, CA, USA) were added to 25 mL of TBE buffer/agarose, and 3 µL of each sample were loaded to the gel. Electrophoresis was done at 90V for 40 min using a PowerPac 300 power supply (Biorad, CA, USA). DNA bands were visualized using UV light in an Infinity 115 (Vilber Lourmart, Marné La Vallée, France) gel documentation system with the BioCapt software (Vilber Lourmart, Marné La Vallée, France). To determine the molecular weight, we used AccRuler 100 Bp Plus DNA RTU ladder (Maestrogen, Hsinchu City, Taiwan) which includes band sizes from 3000 bp to 100 bp.

Genomic DNA was isolated from a maximum of 100 mg of the aortic samples using the modified “salt out” method [27 (link)] after cell lysis by Proteinase K (ThermoScientific, Waltham, MA, USA).
The quantity and purity of isolated DNA were examined by standard spectrophotometry using a Nanodrop 2000 spectrophotometer (ThermoScientific, Waltham, MA, USA). DNA integrity was tested on a 0.7% agarose gel (Bio Rad Power Pac 300, Hercules, CA, USA) after visualization using ethidium bromide.

The primers of AGP1, AGP2, SS1, SS2, SS3, SS4, GBSS1, GBSS2, SBE1, SBE2A, SBE2B, ISO1, AMY1, AMY2, AMY3, AMY4, BAM1, BAM2, BAM3, BAM4, BAM5, BAM6, and BAM7 were designed using Primer Premier 5.0 software according to sequences published in the National Center for Biotechnology Information (NCBI). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The reference control was wheat ACTIN gene. The specificity of the primers was tested and the PCR conditions were optimized using gradient PCR and agarose gel electrophoresis (Bio Rad, Power Pac 300, USA). The primer sequences are presented in Supplementary Table 1.

Gel electrophoresis was carried out on polyacrylamide (with an acrylamide/Bis ratio of 30:1) slab gels (60 × 80 × 1 mm³) using the discontinuous buffer system of Laemmli. The separating gel of 10% polyacrylamide was underlain with 4% stacking gel, and the running buffer consisted of 0.025 M Tris, 0.2 M glycine, and 0.1% SDS. Gels were run in a Mini-Protein II dual slab cell (Bio-Rad) at a constant current of 10 mA per slab gel using a Power PAC 300 (Bio-Rad). Samples were mixed 1:1 (vol/vol) with sample buffer (0.5 M Tris·HCl, pH 6.8, 2% SDS, 10% glycerol, 4% 2-mercaptoethanol, and 0.05% bromophenol blue), and the samples (normalized for protein content before they were loaded to 20 μg of protein) were resolved.

Active RP-HPLC protein fractions were subjected to SDS-PAGE using Laemmli method [21 ], prior to MALDI-TOF/TOF MS analysis. The protein separation was done on 16% polyacrylamide Tris/HCl gels. Prestained Protein Marker, broad range 6–175 kDa (Bio-Rad), was used as a molecular weight marker. Samples were boiled at 90°C for 5 minutes prior to electrophoresis. The electrophoresis was carried out using Bio-Rad Power Pac 300 at a constant voltage of 60 V for the first 15 minutes, followed by gradual increase to 80–100 V. The gels were then stained with Coomassie Brilliant Blue R-250 and documented. In order to improve visualisation of faint protein bands, MS compatible silver staining method was employed. The gels' images were captured for identifications. Excised protein bands from silver-stained SDS-PAGE gel were subjected to in-gel tryptic digestion and later prepared for MALDI-TOF/TOF MS analysis. Intact RP-HPLC protein fractions were also subjected to in-solution tryptic digestion and MALDI-TOF/TOF MS.