PMC:6599329 / 31817-33185 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/6599329","sourcedb":"PMC","sourceid":"6599329","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6599329","text":"Protein sample preparation\nProtein isolation from cell pellets of different planktonic and biofilm stages was carried out by bead beating as described previously [43]. Briefly, cell pellets were resuspended in PBS (1 ml PBS per gram of cells) and transferred to 2 ml microcentrifuge tubes containing glass beads (0.5 mm) and protease inhibitor (1 mM Phenylmethyl Sulfonyl Fluoride, PMSF) and incubated on ice for 5 min. The tubes were then placed in a Mini Bead beater (BioSpec Products, USA) and subjected to one-minute pulses at 4200 rpm for three times with 1 min interval. The suspensions were centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatant was collected. Protein concentration was estimated by bicinchoninic acid protein assay kit as followed by the manufacturer’s instructions (Thermo Scientific, Massachusetts, United States). Three biological replicates and three technical replicates were used in this experiment. 100 μg of protein (1 μg/μl) from each sample was made up to 100 μl with 50 mM Ammonium bicarbonate and subjected to disulfide reduction, alkylation and in-solution tryptic digestion as followed by previous publication [44]. The digested peptide solution was centrifuged at maximum rpm at 4 °C for 12 min and the supernatant was stored at −20°C until the analysis using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).","divisions":[{"label":"title","span":{"begin":0,"end":26}}],"tracks":[{"project":"2_test","denotations":[{"id":"31253082-26025969-12221390","span":{"begin":163,"end":165},"obj":"26025969"},{"id":"31253082-24856245-12221391","span":{"begin":1160,"end":1162},"obj":"24856245"}],"attributes":[{"subj":"31253082-26025969-12221390","pred":"source","obj":"2_test"},{"subj":"31253082-24856245-12221391","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#abec93","default":true}]}]}}