Pageruler plus protein ladder 10 to 250 kda

The electrophoretic analysis (SDS-PAGE) of defatted meals from untreated and roasted flaxseeds was performed under reducing (R) and non-reducing (NR) conditions (i.e., with or without DTT and 2-mercaptoethanol in a sample buffer and reservoir buffer for electrophoresis, respectively) according to the procedure described by Waszkowiak et al. [29 (link)]. The separation was performed in 15% polyacrylamide separating gel. The protein content in each separated sample was 12 μg (it was measured with a 2-D Quant Kit, GE Healthcare Bio-Sciences, Marlborough, MA, USA). Gel images were acquired with an ImageMaster VDS imaging system and analyzed with the ImageMaster 1D Elite v.4.0 programme (Pharmacia Biotech, Vienna, Austria). Semi-quantitative analysis of selected peaks with a PageRuler Plus Protein Ladder 10 to 250 kDa (Thermo Scientific, Waltham, MA, USA) was based on the molecular weight (MW) expressed as kiloDaltons (kDa). Computations were based on the assumption that the area of a single protein band amounted to the percentage ratio of the area of all separated protein bands (Figure 1), which was 100%. The percentage share of protein bands was analyzed statistically.

Trichloroacetic acid (TCA), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) and 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) were purchased from Sigma-Aldrich (Munich, Germany). Bromophenol blue sodium salt (3′,3″,5′,5″-tetrabromophenol sulfophthalein sodium salt), 2-mercaptoethanol, acrylamide, N,N′-methylenebisacrylamide, sodium dodecylsulphate (SDS), and Coomassie Brilliant Blue R-250 were purchased from SERVA Electrophoresis GmbH (Munich, Germany). PageRuler Plus Protein Ladder 10 to 250 kDa was purchased from Thermo Fisher Scientific (Waltham, MA, USA), and 2-D Quant Kit from GE Healthcare Bio-Sciences (Marlborough, MA, USA). Fluorescein sodium salt was purchased from Fluka (Everett, WA, USA). Pronase E (4,000,000 PU/g) was purchased from Merck (Darmstadt, Germany). Other solvents and reagents of analytical (ACS) or HPLC grade were purchased from Sigma-Aldrich, SERVA Electrophoresis (Munich, Germany) or POCH (Gliwice, Poland).