All steps in the extraction until elution were performed at 4°C to avoid
acid degradation of inositol pyrophosphates. First, the titanium dioxide
(TiO2) beads (Titansphere TiO 5 µm; GL Sciences) were
weighed and prepared by washing once in water then once in 1 M perchloric acid
(PA). Generally 4–5 mg of beads was used for each sample. After
centrifuging at 3500g for 1 min, the beads were resuspended in
PA.
Cells were harvested as appropriate and washed in PBS. A small aliquot was
removed for later protein quantification, enabling normalization. The cells were
pelleted and extracted using 800 µl PA (pH 1). After resuspension in the
acid, samples were kept on ice with vortexing for 10 min, then centrifuged at 18
000g for 5 min, at 4°C. The supernatants were
removed into new eppendorfs and TiO2 beads added (4 mg in 50
μl PA). Samples were vortexed briefly then rotated at 4°C for 15
min; the inositol phosphates and other molecules were adsorbed onto the beads at
this point. Beads were pelleted by centrifuging at 3500g for 1
min, and then washed twice in PA with supernatants discarded. To elute, 200
µl 10% ammonium hydroxide (pH 10) was added to the beads. Samples
were vortexed briefly before rotation for 5 min. After centrifuging, the
supernatants (containing the inositol phosphates) were transferred into new
eppendorfs. The elution procedure was repeated on the beads to ensure full
recovery, and the second supernatants added to the first. The samples were then
vacuum evaporated to 50 µl for PAGE or other further analysis.
Alternatively, samples were evaporated until at pH 7 then stored at 4°C
or −20°C.
The protocol used for TiO2 extraction from
Dictyostelium PA extracts, diluted InsP6standards and radioactive 3H-Ins(1,4,5)P3 (PerkinElmer) or
3H-InsP6 (Amersham) was the same as above, except that
the standards were directly added to 1 ml PA. For the radioactive experiments, 5
ml of Ultima Gold (PerkinElmer) scintillation cocktail was added to the
TiO2 eluate and the samples were counted in a
β-counter.
Mouse liver and brain were collected from newborn (P1) pups and rapidly frozen.
PA (2 ml) was added to approximately 0.5 g of tissues, equivalent to one liver
or two brains. The organs were rapidly homogenized in an electric blender and
incubated in ice for 10 min. The samples were centrifuged at more than 15
000g for 15 min and the supernatant used for
TiO2 bead extraction.
acid degradation of inositol pyrophosphates. First, the titanium dioxide
(TiO2) beads (Titansphere TiO 5 µm; GL Sciences) were
weighed and prepared by washing once in water then once in 1 M perchloric acid
(PA). Generally 4–5 mg of beads was used for each sample. After
centrifuging at 3500g for 1 min, the beads were resuspended in
PA.
Cells were harvested as appropriate and washed in PBS. A small aliquot was
removed for later protein quantification, enabling normalization. The cells were
pelleted and extracted using 800 µl PA (pH 1). After resuspension in the
acid, samples were kept on ice with vortexing for 10 min, then centrifuged at 18
000g for 5 min, at 4°C. The supernatants were
removed into new eppendorfs and TiO2 beads added (4 mg in 50
μl PA). Samples were vortexed briefly then rotated at 4°C for 15
min; the inositol phosphates and other molecules were adsorbed onto the beads at
this point. Beads were pelleted by centrifuging at 3500g for 1
min, and then washed twice in PA with supernatants discarded. To elute, 200
µl 10% ammonium hydroxide (pH 10) was added to the beads. Samples
were vortexed briefly before rotation for 5 min. After centrifuging, the
supernatants (containing the inositol phosphates) were transferred into new
eppendorfs. The elution procedure was repeated on the beads to ensure full
recovery, and the second supernatants added to the first. The samples were then
vacuum evaporated to 50 µl for PAGE or other further analysis.
Alternatively, samples were evaporated until at pH 7 then stored at 4°C
or −20°C.
The protocol used for TiO2 extraction from
Dictyostelium PA extracts, diluted InsP6standards and radioactive 3H-Ins(1,4,5)P3 (PerkinElmer) or
3H-InsP6 (Amersham) was the same as above, except that
the standards were directly added to 1 ml PA. For the radioactive experiments, 5
ml of Ultima Gold (PerkinElmer) scintillation cocktail was added to the
TiO2 eluate and the samples were counted in a
β-counter.
Mouse liver and brain were collected from newborn (P1) pups and rapidly frozen.
PA (2 ml) was added to approximately 0.5 g of tissues, equivalent to one liver
or two brains. The organs were rapidly homogenized in an electric blender and
incubated in ice for 10 min. The samples were centrifuged at more than 15
000g for 15 min and the supernatant used for
TiO2 bead extraction.
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