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GNPS - 10142020_Fe_binding_compounds_Keyzers

ABSTRACT: Purified rutin and nicotiflorin and extracts were subjected to iron addition at 500 uM and 1 mM concentrations.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Psychotria Insularum (ncbitaxon:77904)

SUBMITTER: Pieter C. Dorrestein

PROVIDER: MSV000086287 | GNPS | Wed Oct 14 14:28:00 BST 2020

REPOSITORIES: GNPS

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Project description:Approximately 1 x 10^8 GFP-HA-KERP2 expressing Entamoeba histolytica cells were cross-linked in 0.5% formaldehyde (in PBS) for 10 minutes at room temperature. Cross-linking was quenched with 125 mM glycine for 5 minutes. Cells were washed once with PBS and subjected to nuclear fractionation using nuclei isolation buffer (10 mM Hepes-KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 2% NP-40, 1 uM E-64, 0.5 mM PMSF, and 1x cOmplete protease inhibitor cocktail). Nuclear pellets were resuspended in sonication buffer (50 mM Tris-HCl pH 8.0, 140 mM NaCl2, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 5 mM DTT, 1 uM E-64, 0.5 mM PMSF, 1x cOmplete) and sonicated using a Covaris instrument. Sonicated samples were centrifuged at 16000 x g for 20 minutes. A 200 uL aliquot of supernatant was transferred to a 1.5 mL Protein LoBind tube. Remaining sample was stored as input control. For immunoprecipitation, 1 mL of ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 0.5% BSA, 1x cOmplete) containing 50 uL of GFP(ab290)-antibody conjugated Dynabeads was added and rotated at 4 C for 12 hours. Beads were washed five times with low-salt wash buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), followed by two washes with high-salt wash buffer (same as above, but 500 mM NaCl). DNA-protein complexes were eluted with 200 uL of ChIP elution buffer (10 mM Tris-HCl pH 8.0, 1% SDS, 5 mM EDTA, 300 mM NaCl) at room temperature for 20 minutes. Input samples were thawed and diluted with 190 uL of elution buffer. All samples were incubated at 65 C for 8 hours to reverse cross-links, followed by RNase A treatment (1 uL) at 37 C for 1 hour and Proteinase K digestion (5 uL) at 55 C for 2 hours. DNA was purified by phenol/chloroform extraction with 1 uL glycogen as carrier, followed by ethanol precipitation overnight at -30 C. DNA pellets were washed with 80% ethanol, air-dried, and resuspended in 20 uL TE (ChIP) or 40 uL TE (input). DNA concentrations were quantified using a Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific). Purified DNA was submitted to Rhelixa for ChIP-seq.

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GNPS - 10142020_Fe_binding_compounds_Keyzers

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