Beak mill homogenizer

Day 1 adult N2 worms were bleached, and eggs were allowed to hatch in S-complete by shaking overnight. On the next day, 12,000 L1 worms were seeded in a 15 cm plate containing a total volume of 30 mL S-complete with 6 mg/mL OP50 bacteria, 50 μg/mL carbenicillin, and 0.1 μg/mL amphotericin B. Six mL of 0.6 mM FUDR were added to worms at L4 stage in each plate. 100 μΜ translation inhibitor was added to worms 2 hr after adding FUDR. After 12 hr, worms were transferred into a 15 mL corning tube containing a total volume of 5 mL S-complete with 750 µL 6 mg/mL OP50 bacteria, 0.5 mg/mL puromycin, and 100 μΜ translation inhibitors. After rotating the corning tubes for 4 hr, worms were collected into 2 mL cryotubes by washing them with M9 once and with cold PBS three times. Worms were flash-frozen in liquid nitrogen and subsequently broken with a beak mill homogenizer (Fisherbrand). Protein concentrations were determined by the Bradford protein assay. 50 mg protein from each sample was loaded for western blot analysis using antibodies against puromycin (Millipore, MABE343) and GAPDH (Proteintech, 10494-1-AP). Antibodies were diluted 1:5000 in 5% non-fat milk in TBST.

The Chemicon 20S Proteasome Activity Assay Kit (Cat. No APT280) was used according to the manufacturer’s instructions. In short, 10,000 age-synchronized animals were grown in liquid culture on 10 cm plates. DMSO or inhibitors were added on day 1. On day 5, animals were collected and washed 3× with cold DPBS, then once with 1× assay buffer. Worms were flash-frozen in liquid nitrogen. After three biological replicates were harvested in this way, the frozen animals were broken open with a beak mill homogenizer (Fisherbrand). Protein concentrations were determined by the Bradford protein assay. 200 μg of sample was loaded with assay mixture into a 96-well plate and incubated for 1 hr at 37°C. Fluorescence was measured on the Tecan Safire II with a 380/460 nm filter set. Positive control: 5 µL of 20S proteasome positive control (Chemicon Part No. 90205). Negative control: N2 lysate treated with 25 mM lactacystin, a 20S proteasome inhibitor (Chemicon Part No. 90208).

The Chemicon 20S Proteasome Activity Assay Kit (Cat. No APT280) was used according to the manufacturer’s instructions. In short, 10,000 age-synchronized animals were grown in liquid culture on 10 cm plates. DMSO or inhibitors were added on day 1. On day 5, animals were collected and washed 3× with cold DPBS, then once with 1× assay buffer. Worms were flash-frozen in liquid nitrogen. After three biological replicates were harvested in this way, the frozen animals were broken open with a beak mill homogenizer (Fisherbrand). Protein concentrations were determined by the Bradford protein assay. 200 μg of sample was loaded with assay mixture into a 96-well plate and incubated for 1 hr at 37°C. Fluorescence was measured on the Tecan Safire II with a 380/460 nm filter set. Positive control: 5 µL of 20S proteasome positive control (Chemicon Part No. 90205). Negative control: N2 lysate treated with 25 mM lactacystin, a 20S proteasome inhibitor (Chemicon Part No. 90208).