Owl dual gel vertical electrophoresis systems

The polymorphism of avenin peptides was studied in the condition of the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli [22 (link)] on 14% separating gels and 4.6% stacking gels in Thermo Scientific™ Owl™ Dual-Gel Vertical Electrophoresis Systems (US) units. Extractable proteins were diluted in the ratio 1:1 (v/v) with the sample buffer (0.055 M Tris–HCl, pH 6.8; 2% SDS; 40% glycerol; 1% DTT; and 0.0025% bromophenol blue) and heated at 90 °C for 5 min. After the run, the proteins were fixed for 30 min in 12% trichloroacetic acid and stained for 30 min with Coomassie Brilliant Blue R-250. Molecular weights of the polypeptides were estimated by using the Thermo Scientific™ PageRuler™ Unstained Broad Range Protein Ladder.

Logarithmic phase T. brucei brucei bloodstream parasites were collected by centrifugation at 3500× g rpm, for 30 min at 4 °C (Allegra X-15R, Beckman Coulter, Brea, CA, USA), and washed twice in sterile DPBS 1X pH 7.4. The resulting cell pellets (from 5 × 10⁹ cell/mL inoculum) were lysed by adding 300 µL of RIPA lysis buffer consisting of 50 mM TrisHcl, pH 7.5, 150 mM NaCl, 1% Triton X-100 v/v, and 1% protease inhibitor cocktail. The homogenate was stirred for 30 min at 25 °C followed by 10 freeze (−80 °C)/thaw (42 °C) cycles to maximize lysis and extraction. Proteins extract was centrifuged at 14,000× g rpm (Eppendorf 5418-RG, Eppendorf AG, Hambourg, Germany) for 15 min at 4 °C, and the supernatant was collected and conserved at −80 °C until further use.
The protein concentration of the crude extract was determined by the Bradford method (Bradford protein assay kit 23200) using a bovine serum albumin standard curve [74 (link)]. Protein extraction was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Owl Dual-Gel Vertical Electrophoresis systems, Thermo Scientific, Waltham, MA, USA) [75 (link)]. For this purpose, acrylamide separating and stacking gels were run at approximately 100 to 120 V during 1 h. At the end of the electrophoresis, gels were stained with Coomassie brilliant blue G-250 and destained with distilled water for visualization.