Liver GO activity was measured by the formation of glyoxylate from glycolate in liver tissue lysates as previously described [14 ]. A Kinetex 2.6 µm C18 100 Å LC column (100 × 4.6 mm) (Phenomenex Inc, Torrance, CA) was used at a flow rate of 0.4 ml/min, 20 °C, with UV detection at 320 nm to separate and measure the phenylhydrazone products. The mobile phase contained 0.1 M ammonium acetate, 4% acetonitrile and 4% methanol. Assays were incubated at room temperature in the dark for 15 minutes before injection. HYPDH enzymatic activity in isolated mitochondria was measured as previously described [15 (link)]. Each assay contained 0.06 mg mitochondrial protein. The reaction product 3-OH-P5C was derivatized with o-aminobenzaldehyde (o-AB) and adduct formation monitored continuously at 443 nm over 60 minutes at 37 °C in a Syntek plate reader (Synergy HT Microplate Reader). Each sample was run in duplicate.
Kinetex 2.6 m c18 100 lc column 100 4 6 mm
Manufactured by Phenomenex
The Kinetex 2.6 µm C18 100 Å LC column (100 × 4.6 mm) is a high-performance liquid chromatography (HPLC) column with a particle size of 2.6 μm and a pore size of 100 Å. It is designed for use in analytical separation and purification applications.
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2 protocols using kinetex 2.6 m c18 100 lc column 100 4 6 mm
1
Measuring Liver Glyoxylate and HYPDH Activity
2
Extraction and Quantification of Tocopherols from Seeds
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Arabidopsis seeds were harvested and collected in 2 ml Eppendorf tubes, a few pieces of Drierite were added (cat # 238988, Sigma-Aldrich) and the tubes capped and kept at room temperature for at least 4 weeks before weighing and extraction. For corn, ears of field-grown maize were harvested and air-dried at 37 °C for 3 days, hand shelled, ground to powder using IKA tube mill (cat # 0004180001, IKA) under the setting of: speed 25,000 rpm, total time: 2 min, grinding interval time: 5 s with at least 30 s paused between grindings. Details for extracting tocopherols from Arabidopsis and maize seeds are listed as short protocols following the methods section. HPLC separation and quantification were performed using a Kinetex® 2.6 µm C18 100 Å, LC Column 100 × 4.6 mm (cat # 00D-4462-E0, Phenomenex) with the following conditions. Gradient (Time, B %): 0–3 min, 100; 3–5 min, 85; 5–10 min, 30; 10–11.2 min, 0; 11.2–13, 100. Oven temperature was 40 °C and FLD (Fluorescence detector; Shimadzu model, RF-20A, cat# 228-45147-42) settings were Excitation Wavelength 290 nm and Emission Wavelength 330 nm with attenuation set to low sensitivity. Solvent B: 85:15:0.1 v/v/v Acetonitrile/Water/Triethylamine; Solvent A: 100% Ethyl Acetate; Pump flow: 2 ml/min.
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