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MB Sample ID: SA301642
Local Sample ID: | 539_06 |
Subject ID: | SU002917 |
Subject Type: | Plant |
Subject Species: | Arabidopsis thaliana |
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Sample Preparation:
Sampleprep ID: | SP002923 |
Sampleprep Summary: | 3’,5’,5’,7’,7’,7’-d6-labelled ABA and 17,17-d2-labelled GA12 were purchased from OlChemIm, Czech Republic. PA and 7’,7’,7’-d3-PA were gifts from Professor Eiji Nambara (University of Toronto, Canada). Preparations of E. coli cell lysates were incubated in a total volume of 100 µl containing 100 mM Tris-HCl, pH 7.0 at 30°C for 16 h with 2-oxoglutarate and ascorbate (100mM each, final concentrations), FeSO4 (0.5 mM), catalase (1mg/ml), and the substrates (5 µl in methanol for ABA, 3’,5’,5’,7’,7’,7’-d6-labeled ABA (500 ng), PA (500 ng), and 7’,7’,7’-d3-labeled PA (50 ng), and 2 µl in methanol per 5 ng 17,17-d2-labelled GA12. Variations to the standard incubation conditions are indicated in the individual experiments. Incubation products were extracted and analyzed by reverse-phase HPLC as described previously12 using gradients of increasing methanol in water, containing 1% acetic acid, at 1 ml.min-1 as follows: 50% methanol, followed by five 0.5-min steps to 57.4%, 60.6%, 61.8%, 62.3%, 62.5%, one 5-min step to 62.7%, one 1-min step to 63.2% and seven 2-min steps to 64.3%, 67.2%, 70.5%, 74.9%, 81%, 89% and 100% methanol. PA and compound A eluted between 3 and 6 min, and ABA between 6 and 9 min. The dried HPLC-fractions were redissolved in 100 µl methanol and methylated with 100 µl ethereal diazomethane. |