PMC:4236617 / 10266-12382 JSONTXT

{"project":"2_test","denotations":[{"id":"25070625-10675692-12189973","span":{"begin":615,"end":617},"obj":"10675692"}],"text":"Purification of porcine intestinal mucus\nThe 8-week old pig used for mucus isolation was housed in a piggery of MTT Agrifood Research (Finland), treated in strict accordance with the recommendations of the Finnish Ministry of Agriculture and Forestry (Directive 2013–497) and EEC (Directive 86/609/EEC) for the care and use of animals in research, and sacrificed by bolt gun. As the pig used in this study was not specifically included in any experimental protocol on living animals before slaughtering, there was no ethical requirement for collecting mucus samples. The mucus isolation protocol was modified from [34]. Briefly, the small intestine was opened longitudinally and washed with cold phosphate-buffered saline (PBS) with 0.1 mM phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor (PBS-PMSF). Mucus was collected by gentle scraping into PBS-PMSF, centrifuged (17,000 g, 1 hour, +4°C) to remove cells and insoluble material, and the supernatant was homogenized in a domestic blender. The homogenate was concentrated in a Centricon Plus-70 filter unit (molecular weight cutoff 10,000), clarified by centrifugation (17,000 g, 30 min, +4°C), filtered twice through a glass fibre filter (GE Healthcare, Little Chalfont, UK) and once through a 0.8 μm cellulose acetate filter (Sartorius, Goettingen, Germany) and purified by gel filtration chromatography at +4°C in a Sephacryl S-200 HiPrep 16/60 column (GE Healthcare, Little Chalfont, UK) at a flow rate of 1.8 ml/min with PBS as the eluent, monitoring the A280nm values of 5 ml fractions. The protein-containing fractions were dialyzed against water and assayed for total protein by the method of Bradford (Bio-Rad Protein Assay, Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as a standard, and for glycoproteins using the Crypton™ Glycoprotein Staining Kit (Thermo Scientific, Waltham, MA), with porcine gastric mucins (Sigma), horseradish peroxidase (HRP) and soybean trypsin inhibitor (Thermo Scientific, Waltham, MA) as standards. The void volume fractions with a high glycoprotein content were pooled, lyophilized and stored at -20°C."}

{"project":"MicrobeTaxon","denotations":[{"id":"T77","span":{"begin":16,"end":23},"obj":"9823"},{"id":"T78","span":{"begin":1874,"end":1881},"obj":"9823"},{"id":"T79","span":{"begin":56,"end":59},"obj":"9823"},{"id":"T80","span":{"begin":383,"end":386},"obj":"9823"},{"id":"T81","span":{"begin":1726,"end":1732},"obj":"9913"},{"id":"T82","span":{"begin":1939,"end":1946},"obj":"3847"},{"id":"T83","span":{"begin":1906,"end":1917},"obj":"3704"}],"namespaces":[{"prefix":"_base","uri":"http://purl.bioontology.org/ontology/NCBITAXON/"}],"text":"Purification of porcine intestinal mucus\nThe 8-week old pig used for mucus isolation was housed in a piggery of MTT Agrifood Research (Finland), treated in strict accordance with the recommendations of the Finnish Ministry of Agriculture and Forestry (Directive 2013–497) and EEC (Directive 86/609/EEC) for the care and use of animals in research, and sacrificed by bolt gun. As the pig used in this study was not specifically included in any experimental protocol on living animals before slaughtering, there was no ethical requirement for collecting mucus samples. The mucus isolation protocol was modified from [34]. Briefly, the small intestine was opened longitudinally and washed with cold phosphate-buffered saline (PBS) with 0.1 mM phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor (PBS-PMSF). Mucus was collected by gentle scraping into PBS-PMSF, centrifuged (17,000 g, 1 hour, +4°C) to remove cells and insoluble material, and the supernatant was homogenized in a domestic blender. The homogenate was concentrated in a Centricon Plus-70 filter unit (molecular weight cutoff 10,000), clarified by centrifugation (17,000 g, 30 min, +4°C), filtered twice through a glass fibre filter (GE Healthcare, Little Chalfont, UK) and once through a 0.8 μm cellulose acetate filter (Sartorius, Goettingen, Germany) and purified by gel filtration chromatography at +4°C in a Sephacryl S-200 HiPrep 16/60 column (GE Healthcare, Little Chalfont, UK) at a flow rate of 1.8 ml/min with PBS as the eluent, monitoring the A280nm values of 5 ml fractions. The protein-containing fractions were dialyzed against water and assayed for total protein by the method of Bradford (Bio-Rad Protein Assay, Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as a standard, and for glycoproteins using the Crypton™ Glycoprotein Staining Kit (Thermo Scientific, Waltham, MA), with porcine gastric mucins (Sigma), horseradish peroxidase (HRP) and soybean trypsin inhibitor (Thermo Scientific, Waltham, MA) as standards. The void volume fractions with a high glycoprotein content were pooled, lyophilized and stored at -20°C."}