Coomassie blue protein assay bradford kit

Small intestinal IAP specific activity (as it relates to protein) was measured as previously described [15 (link)] and expressed as picomoles pNPP hydrolyzed/min/μg of protein. Briefly, thoroughly washed duodenal tissues were homogenized with lysis buffer (150 mM NaCl, 10 mM Tris·HCl, pH 7.5, 1% sodium deoxycholate, 1% Nonidet P-40, 10 mM EDTA, 0.1% SDS, including protease inhibitor mixture; Sigma) followed by incubation on ice for 30 min. Thereafter, the homogenates were centrifuged twice at 4 °C at 15,000g for 15 min, and the supernatants were collected to determine IAP activity as well as protein concentration. The Coomassie Blue Protein Assay (Bradford) kit from Fisher Scientific was used for protein quantification. For IAP assay, 25 μL of supernatant was mixed with 175 μL phosphatase assay reagent containing 5 mM of p-nitrophenyl phosphate (pNPP) followed by determining optical density at 405 nm. The specific activity of the enzyme was expressed as picomoles pNPP hydrolyzed/min/μg of protein. Protein concentration in a specific sample was determined using the protein assay reagents from Fisher Scientific.

The IAP assay has been previously described (21 (link)). Briefly, an individual stool sample was homogenized in water (10 mg/mL) followed by incubation on ice for 30 minutes. Thereafter, the homogenates were centrifuged twice at 4°C at 15,000g for 15 minutes, and the supernatants were collected to determine IAP activity as well as protein concentration. The Coomassie Blue Protein Assay (Bradford) kit from Fisher Scientific was used for protein quantification. For IAP assay, 25 μL of supernatant was mixed with 175 μL phosphatase assay reagent containing 5 mM of p-nitrophenyl phosphate (pNPP) followed by determining optical density at 405 nm. The specific activity of the enzyme is expressed as picomoles pNPP hydrolyzed/min/μg of protein.