Biorad precision plus protein dual color standards

For each protein-extract quality evaluation, one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1D SDS-PAGE) was carried out according to Laemmli [63 (link)]. Skim milk and protein extract amounting to 20 µg of protein was diluted 1:1 with sample loading solution (0.5M Tris-HCl, pH 6.8, 10% SDS, Glycerol, and Bromophenol Blue), to reach a final volume of 10 µL before being heated at 95 °C for 5 min. The protein samples and skim milk were then run through 5% stacking gel, followed by 12% resolving gel at 90 V, using a Mini-Protean system (Biorad Laboratories, Hercules, CA, USA). Biorad Precision Plus Protein Dual Color Standards (Biorad Laboratories, Hercules, CA, USA) were loaded in the first lane of each gel, to estimate the molecular weight of proteins. The gels were stained by using Coomassie Brilliant Blue (CBB) staining protocol. Gels were stained with CBB staining solution, on a shaker, for 6 h, at room temperature, before being destained with destaining solution (40% methanol and 7% acetic acid in H₂O) for 40 min.

Two SDS-PAGE gels were prepared as follows:
Gel 1: This gel was used for direct detection of the antimicrobial activity against L. monocytogenes FSL – X1 – 0001, S. aureus L1 – 0030, and E. coli O157: H7 and was washed with distilled water after fixation step. Eight microliters of the sample with a final concentration of 5.49 mg/mL and 3 µL of molecular-weight standard (Bio-Rad Precision Plus Protein™ Dual Color Standards – Bio-Rad, USA) ranging from 10 to 250 kDa were loaded to Mini-PROTEAN precast gels for polyacrylamide gel electrophoresis (PAGE) (Bio-Rad Mini-PROTEAN® TGX Stain-Free™ Precast Gels, Bio-Rad, USA). Electrophoresis conditions and protein visualization with Coomassie Blue Staining were conducted according to the following protocol [48 (link)].
Gel 2: The gel was prepared for band visualization in agreement with the standards set by the Cornell Biotechnology Resource Center (BRC) in 12% Bis-Tris gel/ MES buffer system/ colloidal Coomassie stain.