Gradient 4 12 bis tris pre cast gels

Using gradient 4–12% Bis-Tris pre-cast gels (Invitrogen, Barcelona, Spain) according to the manufacturer’s instructions, denaturing gel analyses of biscuit proteins before and after digestion were conducted. The running buffer employed was 2-N-morpholine-ethane sulphonic acid (NuPAGE MES, Invitrogen). As previously described by Olias et al. [21 (link)], before loading, the samples underwent reduction with DTT, and NuPAGE antioxidant was introduced into the upper buffer chamber to avert the re-oxidation of reduced proteins during electrophoresis (Expedeon, Harston, UK) or InstantBlue (Abcam, Cambridge, UK). Protein standards used in gel analyses included Mark12TM (Invitrogen, LC5677, Spain) with proteins in the range of 2.5 to 200 kDa or SeeBlue Plus2 pre-stained standards ranging from 3 to 198 kDa (Thermofisher, Waltham, MA, USA).

Denaturing gel analyses were carried out using gradient 4–12% Bis-Tris pre-cast gels (Invitrogen), according to the manufacturer’s instructions, with 2-N-morpholine-ethane sulphonic acid (NuPAGE MES, Invitrogen, Barcelona, Spain) as running buffer. Gels were stained using Colloidal Blue (Expedeon, Harston, UK). For accurate molecular weight estimation, the unstained protein standard Mark12TM (Invitrogen, LC5677, Barcelona, Spain) was included in the gel with proteins in the range of 2.5 to 200 kDa.