Bio safe coomassie brilliant blue g 250 stain

Gelatin molecular weight distribution was assessed by SDS-PAGE electrophoresis, using 7.5% acrylamide pre-casted gels (7.5% Mini-Protean^® TGX™ precast protein, 10 well, BioRad, Hercules, CA, USA). Samples were heated at 95 °C for 5 min before loading (50 µg of gelatin and GelMA in distilled water), and standard molecular weight markers in the 250–10 KDa range were used (Kaleidoscope™, Precision Plus Protein Standards™, BioRad, Hercules, CA, USA). The electrophoresis was run at 100 V for 40 min and the resulting gel was stained with Bio-Safe Coomassie Brilliant blue G-250 stain (BioRad, Hercules, CA, USA) for 30 min, following the supplier’s instructions.

Peptide samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing and reducing conditions using 4–12% Invitrogen NuPAGE Novex Bis-Tris precast gels (Thermo Fisher Scientific, Loughborough, UK) and the Invitrogen XCell SureLock Mini-Cell (Thermo Fisher Scientific, Loughborough, UK). An aliquot of 10 μL peptide samples was incubated with 4 μL NuPAGE lithium dodecyl sulphate (LDS) sample buffer (Thermo Fisher Scientific) and 2 μL of 500 nM dithiothreitol (DTT) (Fluorochem, Hadfield, UK) for ten minutes at 95 °C and 15 μL of sample mixture loaded per lane and 5 μL of SeeBlue Plus2 pre-stained protein standard (Thermo Fisher Scientific, Loughborough, UK) loaded in at least one lane. The samples were run using 1X 2-morpholinoethanesulfonic acid (MES)-SDS running buffer (Thermo Fisher Scientific, Loughborough, UK). Electrophoresis was performed under a constant current of 125 mA for 40–60 min. The gel was stained using Biosafe Coomassie Brilliant Blue G-250 stain (Biorad, Watford, UK) for 60 min, washed and destained in water overnight and imaged using a Syngene G:box and GeneSnap software (Syngene, Cambridge, UK).