The sample powders were solubilised using in 6M urea and 2% SDS buffer. All samples were diluted to 4 mg/mL (scales TE-124S-OCE, Sartorius AG, Gottingen, Germany), then shaken for 1 min by heavy duty vortex mixer VXHDDG (Ohaus, Parsippany, NJ, USA) at 2500 rpm. The suspensions were then shaken for 1.5 h by environmental shaker incubator ES-20 (Biosan, Ltd., Riga, Latvia) at room temperature. The suspensions passed 15 min centrifugation at G-force 2300 (centrifuge CM-6MT, Elmi Ltd., Riga, Latvia). The resulting supernatants of protein samples were collected and frozen.
Analyses were performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions using Agilent Bioanalyzer 2100 capillary electrophoresis system (Agilent, Santa Clara, CA, USA) with Agilent Protein 230 Kit (14–230 kDa sizing range). Briefly, aliquots of 4 µL unfrozen protein samples were mixed with 2 µL DDT denaturing solution, prepared according Agilent protocols (3.5 Vol, -% of 1M DTT), spined for 15 s and then heated at 95 °C for 5 min, cooled down and diluted to 90 µL with deionized water. Ladder, Gel-Dye mix and destaining solution were prepared and loaded according the Agilent assay protein protocols for Bioanalyzer 2100.
Analyses were performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions using Agilent Bioanalyzer 2100 capillary electrophoresis system (Agilent, Santa Clara, CA, USA) with Agilent Protein 230 Kit (14–230 kDa sizing range). Briefly, aliquots of 4 µL unfrozen protein samples were mixed with 2 µL DDT denaturing solution, prepared according Agilent protocols (3.5 Vol, -% of 1M DTT), spined for 15 s and then heated at 95 °C for 5 min, cooled down and diluted to 90 µL with deionized water. Ladder, Gel-Dye mix and destaining solution were prepared and loaded according the Agilent assay protein protocols for Bioanalyzer 2100.