Bis acrylamide

Acid urea–polyacrylamide gel electrophoresis (AU-PAGE) Western blot analysis was performed on small intestinal tissue and luminal fluid as previously described.^{19 (link),24} Briefly, small intestinal ileal specimens (tissue and luminal aspirate fluid) were homogenized with a Brinkmann Polytron homogenizer in ice-cold 20% acetic acid (1 : 10 w/v) that contained 1 : 100 v/v Protease Inhibitor Cocktail III. The resulting suspension was stirred overnight at 4 °C and clarified the following day by ultracentrifugation (110 000g × 30 min, 4 °C). Aliquots of these specimens were diluted with 0.5 volume of loading buffer (9 M urea, 5% v/v acetic acid, 0.1 mg mL^–1 methyl green (Sigma)) and then resolved on polyacrylamide gels (12.5% acrylamide/2% bis-acrylamide (Roche, Indianapolis, IN), 8 M deionized urea (Sigma), and 5% v/v acetic acid). Samples were run toward the cathode (reverse typical polarity) at 130 volts in 5% v/v acetic acid running buffer until the methyl green indicator dye reached the bottom of the gel (typically ≈1.5 h). Proteins were then transferred from the gels to Immobilon PSQ PVDF membranes in 5% v/v acetic acid using a semi-dry transfer apparatus (Fisher Scientific, Pittsburgh, PA) at 1.5 mA cm^–2 toward the cathode for 20 min. Each membrane was then fixed, washed, blocked, and probed as described for the dot blot analysis.