By Gregory A. Tucker, Jeremy A. Roberts
Confirmed investigators from all over the world describe in step by step aspect their top concepts for the research of plant hormones and their regulatory actions. those state of the art equipment comprise modern ways to selecting the biosynthetic pathways of plant hormones, tracking their degrees, characterizing the receptors with which they have interaction, and reading the signaling platforms wherein they exert their results. finished and completely specified for reproducible laboratory good fortune, Plant Hormone Protocols bargains plant biologists an crucial compendium of cutting-edge strongest equipment and techniques to learning plant hormones, their law, and their actions.
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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Liquid nitrogen. –70 or –20°C freezer. 80% Methanol (HPLC grade). , Andover, MA) see also Note 1. Mortar and pestle. MeOH-resistant centrifuge tube or, for small sample volumes, an Eppendorf microtube. High-speed centrifuge (Centrikon T-124, Kontron, Milan, Italy) or scintered glass filter. For small amounts an Eppendorf centrifuge is appropriate. C18 cartridge. Distilled water. 0 pH indicator strips. 50% MeOH (HPLC grade). DEAE-Sephadex A25 cartridge.
5. Add the 13C6-IAA internal tracer. The amount used (10–1000 pmol) depends on the amount of starting material. The exact amount added should be registrated. 6. IAA is extracted overnight from the tissue at –20°C in darkness. 2. Purification of Free IAA 1. Remove cell debris by centrifugation : 24,000g, 4°C, 15 min. For alternatives see Note 7 and 8. 56 Prinsen et al. 2. Pass the supernatant at 80% MeOH over a C18 cartridge to remove chlorophylls. 3. Rinse the column after application of the sample with an additional 10 mL of 80% MeOH.
Grinding of plant tissue: Micropestles and microtubes or prechilled mortar and pestle. 3. 5 g/L citric acid monohydrate (Baker) and 100 mg/L butylated hydroxytoluene (BHT) (Calbiochem). ) 4. Disposable 14 mL (17 × 100 mm) polypropylene test tubes. 2. Sample Clean-Up 1. 5% extracting methanol. 5% extracting methanol by adding distilled water. 5 mL water. 2. Sep-Pak C18 cartridges (Millipore). Our laboratory does not reuse the C18 cartridges. 3. Disposable syringes (3 mL). 4. Disposable 14-mL (17 × 100 mm) polypropylene test tubes.