Commercial wheat gliadin (Sigma-Aldrich, St. Louis, MO, USA) was subjected to pepsin (0.1 M HCl, pH 1.8) (Sigma-Aldrich, St. Louis, MO, USA) and then trypsin (pH 7.8) (Sigma-Aldrich, St. Louis, MO, USA) digestion at 37 °C for 4 h with vigorous agitation. The pH was adjusted to 4.5 resulting in a precipitate, which was removed by centrifugation. N-tosyl-l-phenylalanine chlorome-thyl ketone and N- α-tosyl-l-lysine chloromethyl ketone hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) were used to inhibit the residual enzymatic activity. The solution was dialyzed, filtered and lyophilized. The powder was dissolved in sterile water at 50 mg/mL, aliquoted and stored at −80 °C.
N tosyl l phenylalanine chlorome thyl ketone
Manufactured by Merck Group
Sourced in United States
N-tosyl-l-phenylalanine chloromethyl ketone is a laboratory reagent used in biochemical research. It functions as a serine protease inhibitor.
Automatically generated - may contain errors
Lab products found in correlation
Nα tosyl l lysine chloromethyl ketone hydrochloride, by Merck Group (2 mentions)
Pepsin, by Merck Group (2 mentions)
Trypsin, by Merck Group (1 mentions)
Gliadin, by Merck Group (1 mentions)
Leupeptin hemisulfate, by Merck Group (1 mentions)
Apoprotinin, by Merck Group (1 mentions)
Powergen 125, by Thermo Fisher Scientific (1 mentions)
Pepstatin a, by Merck Group (1 mentions)
Model 550 microplate reader, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
3 protocols using n tosyl l phenylalanine chlorome thyl ketone
1
Wheat Gliadin Enzymatic Digestion
2
Gliadin Digestion Protocol
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The digestion procedure was performed as described by Frazer et al. [58 (link)], with some modifications. Briefly, gliadin (Sigma-Aldrich, St Louis, MO, USA) was digested with pepsin (0.1 M HCl, pH 1.8) (Sigma-Aldrich, St Louis, MO, USA) and then trypsin (pH 7.8) (Sigma-Aldrich, St Louis, MO, USA) at 37 °C for 4 h with vigorous agitation. Adjustment of the pH to 4.5 resulted in a precipitate, which was removed by centrifugation. To inhibit the residual enzymatic activity, both N-tosyl-l-phenylalanine chloromethyl ketone and N- α-tosyl-l-lysine chloromethyl ketone hydrochloride (Sigma-Aldrich, St Louis, MO, USA) were used. After dialysis, PT was sterile filtered and lyophilized. The resulting powder was dissolved in sterile water and stored at −20° C.
3
Porcine Intestinal Cytokine Analysis
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Porcine specific kits for the analyses of TNF-α, IL-6 and IL-10 cytokine contents in the intestinal tissues and plasma samples were purchased from Quantikine (R&D Systems Inc., Minneapolis, MN, USA). Crude tissue homogenate was prepared by homogenizing 0.5 g of jejunal and colonic tissues with a Powergen 125 hand-held tissue homogenizer (Fisher, Pittsburgh, PA, USA) in 1 ml of Hank's buffer of balanced salt mixture without phenol red, calcium and magnesium (HyClone, Logan, UT, USA) but with 1 µl of protease inhibitor cocktail. The cocktail included the following protease inhibitors (mM): 0.02 N-tosyl-L-phenylalanine chloromethyl-ketone; 0.02 N-α-p-tosyl-L-lysine ketone; 0.02 leupeptin hemisulfate; 0.02 apoprotinin; 0.02 pepstatin A and 0.1 PMSF (Sigma/Aldrich). The homogenate was immediately centrifuged at 12000 g at 4 °C and 100 µl of the supernatant (about 0.8 mg protein/sample) was used to initiate the ELISA incubation according to the instructions on the specific kit and absorbance was read by using a BioRad Model 550 Microplate reader (BioRad, Hercules, CA, USA). Protein contents in the supernatant and plasma samples were measured by using a Bio-Rad commercial kit (Lackeyram et al. 2010) .
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