反应详情

Portions of tissues (200-300 mg) were weighed and homogenized in 500 μl ice-cold 0.5 M perchloric acid using 6 mm ceramic beads on a tissue lyzer. Homogenates were centrifuged (10 min at 10,000), and resultant supernatants were neutralized (pH ˜7.4) with 5 M KOH plus 0.5 M triethanolamine hydrochloride with the help of pH paper and centrifuged (10 min at 10,000 g). Most of the procedures were performed at room temperature unlike suggested in the paper. A portion (200 μl) of the neutralized extract was reconstituted in 1 ml of Optiphase supermix and 200 μl of this radioactive mix was counted on a Microbeta unit to access total radioactivity (2-[3H]DG+2-[3H]DG-6-P). Another aliquot (200 μl) was mixed with 200 μl of 0.3 N Ba(OH)2 and 0.3 N ZnSO4. This mixture was centrifuged, and the supernatant was used to determine unphosphorylated 2-[3H]DG levels by using a portion (200 μl) of the neutralized extract was reconstituted in 1 ml of Optiphase supermix and 200 μl of this radioactive mix was counted on a Microbeta unit. Quench corrections were not made at this time. Tissue 2-[3H]DG-6-P was calculated as the difference between total and 2-[3H]DG values.

1. custom(10 min at 10,000)
2. customwith the help of pH paper and centrifuged (10 min at 10,000 g)
3. customwere performed at room temperature
4. extractionA portion (200 μl) of the neutralized extract
5. additionthis radioactive mix
6. additionAnother aliquot (200 μl) was mixed with 200 μl of 0.3 N Ba(OH)2 and 0.3 N ZnSO4
7. extractionneutralized extract
8. additionthis radioactive mix