Figure 1.

TiO₂ purifies inositol phosphates. (a) Flowchart describing the five-step TiO₂ bead extraction procedure. (i) Acidic solution (blue) containing inositol phosphates is incubated with (ii) TiO₂ beads (yellow) for 10 min, before (iii) spinning and washing the beads twice with 1 M PA. Elution occurs by incubation (iv) at basic pH (red) with subsequent spinning and recovering the supernatant (v). This is evaporated (vi) to concentrate and neutralize (grey) the extract. (b) InsP₆ diluted in 1 M PA was purified using TiO₂ and subjected to PAGE with toluidine blue staining. I, input; S, supernatant; W1 and W2, washes; E, eluted. While all the eluted InsP₆ was loaded on the gel only 1/10 (approx. 100 µl) of the S, W1 and W2 fractions were loaded. The acid in these fractions results in slightly compressed and slower migration of the orange G dye. (c) As (b), but using a PA extraction from vegetative state D. discoideum cells as input. These toluidine blue-stained gels are representative of experiments performed at least three times. (d) To calculate the exact percentage of recovery, radioactive ³H-Ins(1,4,5)P₃ and ³H-InsP₆ were subjected to TiO₂ purification. The radioactivity recovered (E) and radioactivity remaining on TiO₂ beads (B) were normalized to the respective radioactive input (I). The graph showing the average ± s.d. (n = 4) is representative of two independent experiments with matching results.