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.