Remaining free thiols in Pias3 were blocked with 20 mM methyl methanethiosulphonate (MMTS) in blocking buffer (25 mM Hepes, pH 7.7, 1 mM EDTA, 0.1 mM neocuproine, 2.5% SDS) at 50°C for 30 min with frequent vortexing followed by separation by non-reducing SDS-PAGE in the dark.
The collected material was then incubated on ice for 30 min with frequent gentle vortexing.
The solution was mixed and incubated at 80°C for 5 min with frequent vigorous shaking.
Vortex at high speed for 40 min, with frequent incubation on ice in every 2-3 minutes.
This was followed by an ID glucose infusion at 3 kcal/min over 100 min (t = 30 130 min) with frequent blood sampling.
The seeds were surface sterilized in a 0.2% HgCl2 solution for 5 min (with frequent shaking), and then thoroughly washed with tap water.
Figure 15 shows the results obtained, where scenarios 1-A, 1-B, 2-A, and 2-B represents 9-min video with moderate bandwidth change, 9-min video with frequent bandwidth change, 21-min video with moderate bandwidth change, and 21-min video with frequent bandwidth change, respectively.
The supernatant was used as the cytoplasmic fraction, and the nuclear pellet was resuspended in 300 μl of ice-cold 20 mmol l−1 HEPES-KOH (pH 7.9) containing 0.4 mol l−1 NaCl, 1 mmol l−1 EDTA, 1 mmol l−1 dithiothreitol, and 1 mmol l−1 PMSF and incubated for 15 min on ice with frequent gentle mixing followed by centrifugation for 5 min at 4°C to remove insoluble materials.
For western blot, oocytes were lysed immediately after being selected with a mouth pipette, in a buffer containing 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 20 mM NaF, 20 mM β-glycerophosphate, 1 mM EDTA, 6 mM EGTA (pH 8.0), 1% NP-40, 1 mM DTT, 5 mM benzamidine, 1 mM PMSF, 250 µM sodium vandadate, 2 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 µg/ml pepstatin, for 20 min on ice with frequent vortexing.
The slices were crushed then melted by incubation at 65°C for 30 min. The melted gel was incubated with 0.5 vol. phenol-cresol, pre-warmed at 65°C, and incubated for 5 min at 65°C with frequent vigorous agitation.
Cells were fixed in culture media with electron microscope grade glutaraldehyde (2.5%) for 30 min at room temperature with frequent inversions.
