Thin Layer Chromatography

1. Prepare TLC plates by coating them with a 1% potassium oxalate solution in water-methanol (1 : 1, v/v). This increases the charge on the plate and thus, the retention of charged compounds. To soak the plate, submerge it in the solution for approx 90 s (see Note 10). Dry the plate in an oven (approx 90-100°C).

2. With a pencil, lightly draw a horizontal line approx 3 cm from the bottom of the plate. Be careful not to scrape up any silica as you are writing on the plate. On the origin lane, mark locations to load each sample, separated by at least 1.5 cm.

3. Load the entire extract at the spot on the origin line. Add the sample in small aliquots (approx 20 iL) and let each spot dry before adding the next one; this will prevent the sample from spreading out too much during loading. Do not let the sample occupy a region greater than 1 cm in diameter while loading, and do not touch the silica with the pipet tip. One may wish to load phosphoinositide standards to identify the phosphorylated products (see Note 11).

4. Develop the plate in the TLC tank using about 100 mL of the chloroformmethanol-water-ammonium hydroxide (60:47:11.3:2, v/v/v/v) solvent system (see Note 5). Allow the solvent migrate to within 1 cm of the top (approx 3 h).

5. Remove the plate from the tank and dry it in the fume hood.

6. Wrap the plate in plastic wrap and detect radioactivity by exposing it to either autoradiography film or a phosphorimager screen.

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