Bioruptor ucd 250hsa

Nuclear proteins were isolated from 10-day-old seedlings as described previously^{38 (link)} with some modifications. Sonication was conducted using a BIORUPTOR^® UCD-250HSA (COSMO BIO CO., LTD.) using power mode H and on/off cycle of 30 s/60 s for a total duration of 12 min on ice in sonication buffer [500 mM LiCl, 0.7% sodium deoxycholate, 1% NP-40, 50 mM HEPES-KOH (pH 7.6), 1 mM EDTA (pH 8.0), working concentration of cOmplete, EDTA-free (Roche), 1 mM Pefablock^® SC (Roche), and 5 mM NaF]. The following antibody and magnet beads were used: mouse anti-GFP (11814460001; Roche), mouse anti-RNAPII Ser2P (MABI0602; MAB Institute), and Dynabeads^™ M-280 sheep anti-mouse IgG (11201D; Thermo Fisher Scientific).

To measure magnesium levels in whole worms, 300 synchronized L4/young adult worms were incubated for 30 min with washing buffer containing 110 mM HNO₃ (semiconductor grade, Wako) and 187 mM NH₃ (ultrapure grade, Kanto Chemical), which corresponds to approximately 300 mOsm/l and pH 7.0–8.0 at room temperature, and were then washed 5 times with washing buffer. Subsequently, worms were boiled at 95°C for 5 min and sonicated using Bioruptor (UCD-250HSA; Cosmo Bio). The homogenates were completely dried by incubation at 98°C, and then subjected to treatment with 100 μl of 40% HNO₃ at 95°C for 2 h. The solution was diluted to 1 ml with ddH₂O and magnesium levels were determined using ICP-MS (7700x; Agilent), according to the manufacturer’s instructions. The magnesium levels were normalized to total protein levels, which were determined using the BCA assay kit (Thermo Scientific). A blank sample was prepared using the same procedure without worms. To measure magnesium levels in the intestine, approximately 300 synchronized L4/young adult worms were cut with a scalpel just behind the pharynx in a drop of washing buffer. The extruded intestine was cut away from the remnants of the body, and the isolated intestines were then washed twice with washing buffer. Magnesium levels were analyzed as described above.