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MB Sample ID: SA347870
Local Sample ID: | WT-IP5-FigS2b |
Subject ID: | SU003309 |
Subject Type: | Plant |
Subject Species: | Arabidopsis thaliana |
Taxonomy ID: | 3702 |
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Sample Preparation:
Sampleprep ID: | SP003316 |
Sampleprep Summary: | The enrichment of InsPs with TiO2 beads and SDS-PAGE assay were performed as described by Wilson et al47. In brief, 1 g total seedlings grown on agar medium for 15 d or 0.1 g dry seeds were ground in liquid nitrogen, suspended in 5 ml of 1 M cold perchloric acid, and kept rotating for 15 min at 4 ℃. After centrifugation at 12,000 g for 10 min at 4 ℃, the supernatant was transferred into a new tube containing 30 mg of TiO2 beads (5 mm Titansphere; GL Sciences, Japan) and kept rotating at 4 ℃ for 20 - 30 min. After centrifugation at 5000 g for 10 min at 4 ℃, the beads were transferred into a new 1.5 ml tube and washed with pre-cold PA for 3-5 times, then eluted with 500 µl of 10 % ammonia solution. The eluate was freeze-dried at -50 ℃ and resuspended with 50 µl of 10 % ammonia solution, and the enriched InsPs was resolved in a 33 % polyacrylamide / Tris–borate–EDTA (TBE) gel and stained with toluidine blue. 10 µM synthetic InsP5 (myo-Inositol-1,3,4,5,6-pentaphosphate ammonium salt, Cayman) and InsP6 (Sichem) were used as the markers and standards. In order to verify the specific components in the eluate, the total eluate was firstly freeze-dried, then dissolved with 100 µl 80 % acetonitrile. |