RP-HPLC analysis was carried out using an Agilent Series 1200 HPLC System with a diode array detector (DAD) and separation across a water:acetonitrile gradient with 0.1% (v/v) TFA. For analysis and quantification of siderophores, a gradient condition of 5–55% acetonitrile over 22 min at 2 ml/min was used on a C18 column (Agilent Zorbax Eclipse XDB-C18 Semi- Preparative; 5 μm particle size; 9.4 × 250 mm) with DAD detection at 254 and 440 nm. Peaks associated with extracellular siderophores were collected and identity confirmed via LC-MS/MS.
Zorbax eclipse xdb c18 semi preparative
Manufactured by Agilent Technologies
The Zorbax Eclipse XDB-C18 Semi-Preparative is a high-performance liquid chromatography (HPLC) column designed for semi-preparative applications. It features a bonded C18 stationary phase and is suitable for the separation and purification of a variety of compounds.
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4 protocols using zorbax eclipse xdb c18 semi preparative
1
Quantification of Microbial Siderophores
2
Gliotoxin Spiking and Detection
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For Gliotoxin spiking experiments, cultures were grown in Sabouraud–dextrose medium for 21 h followed by Gliotoxin addition (2.5 µg ml−1 final) for 3 h (n = 3 biological replicates for all specimens). All strains used are given in electronic supplementary material, table S5. Organic extracts from supernatants were analysed by RP-HPLC with UV detection (Agilent 1200 system), using a C18 RP-HPLC column (Agilent Zorbax Eclipse XDB-C18 Semi-Preparative; 5 µm particle size; 4.6 × 250 mm) at a flow rate of 2 ml min−1. A mobile phase of water and acetonitrile with TFA was used under various gradient conditions. Aspergillus fumigatus wild-type, deletion and complementation strains were grown for 72 h in Czapek Dox medium (unless stated otherwise) followed by organic extraction and LC–MS analysis as previously described [12 (link)]. Supernatants were diluted 1/10 in 0.1% (v/v) formic acid and spin filtered prior to LC–MS analysis (Agilent Ion Trap 6340) to detect BmGT presence. Gliotoxin (purity: 98%) and BmGT (purity: 99%) standards were obtained from Sigma-Aldrich and Enzo Life Sciences, respectively.
3
Comparative Analysis of Gliotoxin Biosynthesis in Aspergillus fumigatus
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A. fumigatus wild-type, DgtmA and gtmA c strains (Dolan et al., 2014) (link) were grown (10 8 conidia/ml) in quadruplicate (Czapek-Dox broth, 200 rpm, 3 d; then static, 25 d 37 C). Culture supernatants and ethyl acetate organic extracts (100 ml) were analysed by RP-HPLC with UV detection (Agilent 1200 system), using a C18 RP-HPLC column (Agilent Zorbax Eclipse XDB-C18 Semi-Preparative; 5 mm particle size; 4.6 Â 250 mm) at a flow rate of 2 ml/min (Fig 1 ).
A mobile phase of water and acetonitrile with 0.1 % (v/v) trifluoroacetic acid, was used under various gradient conditions. For LCeMS analysis, organic extracts were diluted 1/10 in 0.1 % (v/v) formic acid and spin filtered prior to LCeMS analysis (Agilent Ion Trap 6340). Gliotoxin (purity: 98 %) and BmGT (purity: 99 %) standards were obtained from Sigma-Aldrich and Enzo Life Sciences, respectively. Fumagillin, pseurotin A, tryprostatin B and fumitremorgin C were identified based on published m/z ratios, retention times and fragmentation patterns as described previously (O'Keeffe et al., 2014) (link). All data were analysed using built-in GraphPad prism version 5.01 functions, as specified. The level of significance was set at p < 0.05 (*), p < 0.001 (**), and p < 0.0001 (***), unless otherwise stated.
A mobile phase of water and acetonitrile with 0.1 % (v/v) trifluoroacetic acid, was used under various gradient conditions. For LCeMS analysis, organic extracts were diluted 1/10 in 0.1 % (v/v) formic acid and spin filtered prior to LCeMS analysis (Agilent Ion Trap 6340). Gliotoxin (purity: 98 %) and BmGT (purity: 99 %) standards were obtained from Sigma-Aldrich and Enzo Life Sciences, respectively. Fumagillin, pseurotin A, tryprostatin B and fumitremorgin C were identified based on published m/z ratios, retention times and fragmentation patterns as described previously (O'Keeffe et al., 2014) (link). All data were analysed using built-in GraphPad prism version 5.01 functions, as specified. The level of significance was set at p < 0.05 (*), p < 0.001 (**), and p < 0.0001 (***), unless otherwise stated.
4
Siderophore Identification and Purification
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RP-HPLC analysis was carried out using an Agilent Series 1200 HPLC System with a diode array (DAD) and fluorescence (FLD) detector and separation across a water: acetonitrile gradient with 0.1% (v/v) TFA. For analysis and quantification of siderophores in supernatants, gradient conditions of 5-100% acetonitrile over 35 min at 1 ml/min were used on a C 8 column (Agilent Zorbax Eclipse XDB-C8 Analytical; 5 μm particle size; 4.6 × 150 mm) with DAD detection at 254 and 440 nm. Peaks associated with extracellular siderophores were collected by fractionation and identity confirmed via LC-MS. Purification of FSC +Fe was carried out with gradient conditions of 5-55% acetonitrile over 22 min at 2 ml/min on a C 18 column (Agilent Zorbax Eclipse XDB-C18 Semi-Preparative; 5 μm particle size; 9.4 × 250 mm) with DAD detection at 254 and 440 nm. The peak associated with FSC +Fe was collected by repeated fractionation and fractions pooled.
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