Enzyme reaction mixtures (100 μl) consisted of enzyme ConA (2 μM), 50 mM tris-HCl (pH 8.0), 20 mM NaCl, 1 mM histidine, 1 mM tris(2-carboxyethyl)phosphine, 0.1 mM FeSO4, and 1 mM sulfur-donor substrates (cysteine, glutathione, methionine, and N-acetyl cysteine). The reaction mixture was incubated at room temperature for 20 min and quenched by addition of 30 μl of water with 1.0% TFA. An aliquot (20 μl) of the reaction mixture was diluted in 70 μl of H2O and analyzed by HPLC. HPLC analysis was performed using a Hitachi Primade HPLC system equipped with a DAD detector and autoinjector. Aliquots (20 μl) of the sample were injected onto a reversed-phase C18 analytical column (Luna C18 4.6 × 100 mm, Phenomenex) with a gradient starting at 0% mobile phase B (H2O + 0.1% TFA: MeCN) for 5 min and increased until 5% mobile phase B for 20 min at a rate of 0.7 ml/min. The reaction product was purified by HPLC and characterized by NMR (fig. S3C).
Luna c18 4.6 100 mm
Manufactured by Phenomenex
The Luna C18 4.6 × 100 mm is a reversed-phase high-performance liquid chromatography (HPLC) column. It features a C18 stationary phase and dimensions of 4.6 mm internal diameter and 100 mm length.
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Lab products found in correlation
2 protocols using luna c18 4.6 100 mm
1
Enzymatic Sulfur Compound Synthesis
2
Isolation and Identification of Marine Polypropionates
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Chemical extracts were prepared by homogenizing egg ribbons and tissue sections (~10 cm3) of E. diomedea with acetone (5 mL). The homogenate was centrifuged at 3739 × g for 20 min at 4 °C and the supernatant recovered by aspiration. The acetone extract was dried using a rotovap and resuspended in methanol. Extracts were analyzed using a HPLC using a Hitachi Primade HPLC system equipped with a PDA detector and autoinjector. Aliquots (20 µL) were loaded onto a C18 analytical column (Luna C18 4.6 × 100 mm, Phenomenex) with a gradient starting at 30% mobile phase B (H2O + 0.1% TFA: MeCN) and 70% mobile phase A (H2O) for 5 min and increased until 100% mobile phase B for 25 min at a rate of 1.0 mL/min. Purification of the major polypropionate in E. diomedea tissue extract was performed using a semi-preparative HPLC column (Luna 5 u C18 250 × 10 m) at a flowrate of 2.5 mL/min. 1H NMR data were obtained using a Varian 500 (1H 500 MHz) NMR spectrometer equipped with a 3 mm Nalorac MDBG probe, utilizing residual CDCl3 signal for referencing. Natural products, including tridachione, were identified by comparing data to those reported in the literature. A similar procedure was used to isolate and identify products from P. cf. ocellatus.
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