PMC:7795856 / 32775-34206 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7795856","sourcedb":"PMC","sourceid":"7795856","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7795856","text":"4.10. ELISA Quantification of PASylated Tα1\nA 96-well Nunc Maxisorb ELISA plate (ThermoFisher) was coated with 100 µg/mL anti-PAS antibody in PBS at 4 °C overnight. After washing twice with PBS/T, free binding sites were blocked with 3% w/v bovine serum albumin (BSA) in PBS/T at room temperature for 1 h. After washing 3 times with PBS/T, the rat plasma samples were applied in dilution series in PBS/T supplemented with 0.5% (v/v) plasma from an untreated animal (to maintain a constant proportion of rat plasma constituents). In the same manner, a standard curve was prepared using dilution series of purified Tα1-PAS (used for spiking rat plasma) at defined concentrations. After incubation for 1 h at RT, wells were washed 3 times with PBS/T. To detect bound Tα1-PAS, wells were incubated for 1 h with 50 µl of a 1 µg/mL PBS/T solution of the second anti-PAS antibody conjugated with alkaline phosphatase using the Lightning-Link alkaline phosphatase antibody labeling kit (BioTechne, Wiesbaden, Germany). After washing twice with PBS/T and twice with PBS, the enzymatic activity was detected using p-nitrophenyl phosphate (0.5 mg/mL). To this end, the plate was incubated for 20 min at 30 °C, the absorbance was measured at 405 nm using a SpectraMax M5e microtiter plate reader (Molecular Devices, Sunnyvale, CA, USA) and the Tα1-PAS concentrations in rat plasma samples were quantified by comparison with the standard curve.","divisions":[{"label":"title","span":{"begin":0,"end":43}}],"tracks":[{"project":"LitCovid-PubTator","denotations":[{"id":"580","span":{"begin":248,"end":261},"obj":"Gene"},{"id":"581","span":{"begin":613,"end":616},"obj":"Gene"},{"id":"582","span":{"begin":764,"end":767},"obj":"Gene"},{"id":"583","span":{"begin":889,"end":909},"obj":"Gene"},{"id":"584","span":{"begin":935,"end":955},"obj":"Gene"},{"id":"585","span":{"begin":1332,"end":1335},"obj":"Gene"},{"id":"586","span":{"begin":344,"end":347},"obj":"Species"},{"id":"587","span":{"begin":503,"end":506},"obj":"Species"},{"id":"588","span":{"begin":639,"end":642},"obj":"Species"},{"id":"589","span":{"begin":1358,"end":1361},"obj":"Species"},{"id":"590","span":{"begin":1104,"end":1127},"obj":"Chemical"},{"id":"591","span":{"begin":823,"end":829},"obj":"Disease"}],"attributes":[{"id":"A580","pred":"tao:has_database_id","subj":"580","obj":"Gene:213"},{"id":"A581","pred":"tao:has_database_id","subj":"581","obj":"Gene:50719"},{"id":"A582","pred":"tao:has_database_id","subj":"582","obj":"Gene:50719"},{"id":"A583","pred":"tao:has_database_id","subj":"583","obj":"Gene:16870"},{"id":"A584","pred":"tao:has_database_id","subj":"584","obj":"Gene:16870"},{"id":"A585","pred":"tao:has_database_id","subj":"585","obj":"Gene:50719"},{"id":"A586","pred":"tao:has_database_id","subj":"586","obj":"Tax:10116"},{"id":"A587","pred":"tao:has_database_id","subj":"587","obj":"Tax:10116"},{"id":"A588","pred":"tao:has_database_id","subj":"588","obj":"Tax:10116"},{"id":"A589","pred":"tao:has_database_id","subj":"589","obj":"Tax:10116"},{"id":"A590","pred":"tao:has_database_id","subj":"590","obj":"MESH:C008644"},{"id":"A591","pred":"tao:has_database_id","subj":"591","obj":"MESH:D011535"},{"subj":"580","pred":"source","obj":"LitCovid-PubTator"},{"subj":"581","pred":"source","obj":"LitCovid-PubTator"},{"subj":"582","pred":"source","obj":"LitCovid-PubTator"},{"subj":"583","pred":"source","obj":"LitCovid-PubTator"},{"subj":"584","pred":"source","obj":"LitCovid-PubTator"},{"subj":"585","pred":"source","obj":"LitCovid-PubTator"},{"subj":"586","pred":"source","obj":"LitCovid-PubTator"},{"subj":"587","pred":"source","obj":"LitCovid-PubTator"},{"subj":"588","pred":"source","obj":"LitCovid-PubTator"},{"subj":"589","pred":"source","obj":"LitCovid-PubTator"},{"subj":"590","pred":"source","obj":"LitCovid-PubTator"},{"subj":"591","pred":"source","obj":"LitCovid-PubTator"}],"namespaces":[{"prefix":"Tax","uri":"https://www.ncbi.nlm.nih.gov/taxonomy/"},{"prefix":"MESH","uri":"https://id.nlm.nih.gov/mesh/"},{"prefix":"Gene","uri":"https://www.ncbi.nlm.nih.gov/gene/"},{"prefix":"CVCL","uri":"https://web.expasy.org/cellosaurus/CVCL_"}]},{"project":"LitCovid-sentences","denotations":[{"id":"T199","span":{"begin":0,"end":5},"obj":"Sentence"},{"id":"T200","span":{"begin":6,"end":43},"obj":"Sentence"},{"id":"T201","span":{"begin":44,"end":164},"obj":"Sentence"},{"id":"T202","span":{"begin":165,"end":305},"obj":"Sentence"},{"id":"T203","span":{"begin":306,"end":528},"obj":"Sentence"},{"id":"T204","span":{"begin":529,"end":677},"obj":"Sentence"},{"id":"T205","span":{"begin":678,"end":747},"obj":"Sentence"},{"id":"T206","span":{"begin":748,"end":1010},"obj":"Sentence"},{"id":"T207","span":{"begin":1011,"end":1140},"obj":"Sentence"},{"id":"T208","span":{"begin":1141,"end":1431},"obj":"Sentence"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/ontology/tao.owl#"}],"attributes":[{"subj":"T199","pred":"source","obj":"LitCovid-sentences"},{"subj":"T200","pred":"source","obj":"LitCovid-sentences"},{"subj":"T201","pred":"source","obj":"LitCovid-sentences"},{"subj":"T202","pred":"source","obj":"LitCovid-sentences"},{"subj":"T203","pred":"source","obj":"LitCovid-sentences"},{"subj":"T204","pred":"source","obj":"LitCovid-sentences"},{"subj":"T205","pred":"source","obj":"LitCovid-sentences"},{"subj":"T206","pred":"source","obj":"LitCovid-sentences"},{"subj":"T207","pred":"source","obj":"LitCovid-sentences"},{"subj":"T208","pred":"source","obj":"LitCovid-sentences"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"LitCovid-PubTator","color":"#93ecd8","default":true},{"id":"LitCovid-sentences","color":"#ecbe93"}]}]}}