Novex see blue plus 2

Sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) was carried out as previously described 22 to study the extent of protein digestion. Samples were denatured at 95 o C for 5 min. and were then loaded to a 15% acrylamide and 0.4% bisacrylamide gel for the samples obtained after complete GI digestion, or to a 10% acrylamide/0.3% bisacrylamide gel for the digesta obtained following only gastric digestion. The running time was 30 min. at 80 V followed by 90 min. at 100 V. Protein marker was Invitrogen novex see blue plus 2 (Invitrogen, Carlsbad, CA). After each run, fixation/staining was done with methanol/acetic acid/Coomassie brilliant blue R (50%, 10%, 0.1%) for 30 minutes, and destained in methanol/glacial acetic acid/water (30%, 10%, 60%, 3 h). The gels were scanned by a Kodak Gel Logic 2200 imaging system (Kodak, Rochester, NY).
Free fatty acid determination: Lipid hydrolysis was evaluated by measuring the amount of free fatty acids (FFAs) released in the digesta after GI digestion. This was determined by Cayman's Free Fatty Acid Fluorometric Assay (Cayman Chemical, Art. No. 700310, Ann Arbor, MI) according to the manufacturer's protocol.

The amount of free fatty acids in the digesta after GI digestion was estimated as described previously (Corte-Real, Desmarchelier, Borel, Richling, Hoffmann, & Bohn, 2018) , based on the titration against sodium hydroxide (NaOH 0.1 M), using phenolphthalein as an indicator.
Sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) was carried out to study completeness of protein digestion. Samples were denatured at 95 o C for 5 min. in Lämmli buffer.
About 22 µg of protein per pocket was loaded to a 15% acrylamide and 0.4% bisacrylamide gel (gastro-intestinal digestion) and 45 g of protein per pocket was loaded to a 10% acrylamide/0.3% bisacrylamide gel (gastric digestion). Marker was Invitrogen novex see blue plus 2 (Invitrogen, Carlsbad, CA). Gel was run at 80 V (30 min.) plus 100 V (90 min.). Running buffer contained SDS (0.1%), glycine (190 mM), tris-HCl (25 mM). Fixation/staining was done with methanol/acetic acid/coomassie brilliant blue R (50%, 10%, 0.1%) (30 minutes), destaining in methanol/glacial acetic acid/water (30%, 10%, 60%, 3 h). Pictures were taken in a Kodak Gel Logic 2200 Imaging System (Kodak, Rochester, NY).