The 100 μl assay solution containing 100 mM Tris-HCl (pH 7.5), 50 pmol CmFUM, and 1 M disodium fumarate was incubated at 52°C for 24 h. Thereafter, HCl was added to the assay solution to be 100 mM. The sample is analyzed by HPLC using an LC-2000Plus System (JASCO, Tokyo, Japan). Organic acids were quantified using 0.2 mM bromothymol blue in 15 mM sodium phosphate buffer and peaks were detected at 445 nm, as described previously (Osanai et al., 2015 (link)).
Lc 2000plus system
Manufactured by Jasco
Sourced in Japan
The LC-2000Plus System is a high-performance liquid chromatography (HPLC) instrument designed for analytical and preparative applications. It features a modular design, allowing for the integration of various components, including pumps, autosamplers, and detectors, to create a customized solution for specific laboratory needs. The system provides precise control of solvent delivery, sample injection, and data acquisition, enabling reliable and accurate analysis of a wide range of samples.
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7 protocols using lc 2000plus system
1
Enzymatic Assay of CmFUM Activity
2
Polymerizable Oligonucleotide Synthesis and Purification
3
Enzymatic Assay for Fumarate
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The 500 μl assay solution contains 100 mM Tris-HCl (pH 7.5), 500 pmol CmFUM, and 200 mM disodium fumarate. The assay solution before adding fumarate was pre-incubated for 5 min at 52°C. Thereafter, fumarate which was also pre-incubated for 5 min at 52°C was added to the assay solution and the reaction was started at 52°C. After the reaction for 5, 10, 20, 30, 40, 50, and 60 min, 50 μl of the assay solution was collected and the reaction was stopped by 100 mM HCl. The samples were analyzed by high-performance liquid chromatography (HPLC) using an LC-2000Plus System (JASCO, Tokyo, Japan). Organic acids were quantified using 0.2 mM bromothymol blue in 15 mM sodium phosphate buffer; peaks were detected at 445 nm, as described previously (Osanai et al., 2015 (link)).
4
Quantification of Organic Acids by HPLC
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The freeze-dried supernatants were reconstituted by dissolving in 100 μL of filtered 3 mM perchloric acid and analyzed by HPLC using an LC-2000Plus system (JASCO, Tokyo, Japan) equipped with two RSpak KC-811 columns (Showa Denko, Tokyo, Japan). Quantification of organic acids was achieved using a solution containing 0.2 mM bromothymol blue in 15 mM sodium phosphate buffer, with peak detection occurring at 445 nm. The column was maintained at a temperature of 60°C, and the flow rates were 0.7 mL/min for the 9 mM perchloric acid and 1.2 mL/min for the 0.2 mM bromothymol blue solution. To calibrate the measurements, standard powders of succinate, malate, fumarate, lactate, and acetate were employed, all of which were obtained from Fujifilm Wako Chemicals. Citrate powders were purchased from Nacalai Tesque, INC. (Kyoto, Japan).
5
HPLC Analysis of Organic Acids
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Organic acids were analyzed as described previously (Osanai et al., 2015 (link)). Freeze-dried supernatants were resolved in 100 μL of filtered 3 mM perchloric acid. The resolved samples were analyzed by HPLC using an LC-2000Plus System (JASCO, Tokyo, Japan) with a photodiode array detector and two RSpak KC-811 columns (Showa Denko, Tokyo, Japan). Organic acids were quantified using 0.2 mM bromothymol blue in 15 mM sodium phosphate buffer; peaks were detected at 445 nm. The temperature of the column was 60°C, and flow rates of 3 mM perchloric acid and 0.2 mM bromothymol blue solutions were 0.7 and 1.2 mL/min, respectively.
6
Quantitative Analysis of Organic Acids
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Freeze-dried supernatants were dissolved in 100 μL of filtered 3 mM perchloric acid. The dissolved samples were analyzed by HPLC using a LC-2000Plus Systems (JASCO, Tokyo, Japan) with a photodiode array detector and two RSpak KC-811 columns (Showa Denko, Tokyo, Japan). Organic acids were quantified with 0.2 mM bromothymol blue in 15 mM sodium phosphate buffer; peaks were detected at 445 nm. The column temperature was 60 °C, and the flow rates of 3 mM perchloric acid and 0.2 mM bromothymol blue solutions were 1.0 mL/min and 1.5 mL/min, respectively. Powders of citrate, succinate, lactate, and sodium acetate were used as standards and purchased from Wako Pure Chemicals Co. Ltd (Osaka, Japan).
7
HPLC Analysis of Organic Acids
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Freeze-dried supernatants were resolved in 100 μL of filtered 3 mM perchloric acid. The resolved samples were analyzed by HPLC using a LC-2000Plus Systems (JASCO, Tokyo, Japan) with a photodiode array detector and two RSpak KC-811 columns (Showa Denko, Tokyo, Japan). Organic acids were quantified with 0.2 mM bromothymol blue in 15 mM sodium phosphate buffer; peaks were detected at 445 nm. The column temperature was 60°C, and the flow rates of 3 mM perchloric acid and 0.2 mM bromothymol blue solutions were 1.0 and 1.5 mL/min, respectively.
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