0.22 μm cellulose acetate membrane

We modified the filter-trap analysis as described previously [26 (link)]. Briefly, phosphorylated or non-phosphorylated peptides were diluted with TBS (Tris–HCl, pH 7.5, 150 mM NaCl), and applied to a 0.22-μm cellulose acetate membrane (Millipore) on a slot blot apparatus (Bio-Rad, Hercules, CA, USA) using a vacuum manifold. After washing with TBS containing 0.1% Triton X-100, the membrane was incubated with anti-phosphorylated p62 and pan-p62 antibodies and detected using the ECL detection system as described above.

Lake, spring, and river waters, collected in the Upper Silesian region (Poland), were filtered through a 0.22-μm cellulose acetate membrane (Millipore), and after acidification with HNO₃ stored without access of light at 4 °C. The artificial sea water was prepared by dissolving 21.03 g NaCl, 3.52 g Na₂SO₄, 0.61 g KCl, 0.088 g KBr, 0.034 g Na₂B₄O₇ ∙ 10H₂O, 9.50 g MgCl₂ ∙ 6H₂O, 1.32 g CaCl₂ ∙ 2H₂O, 0.02 g SrCl₂ ∙ 6 H₂O, and 0.02 g NaHCO₃ in 1 L of high-purity water [27 (link)].

Mother culture was prepared by inoculating a loopful of Marinobacter sp. EMB8 from the slant, into 25 mL of medium (A) containing (g/L) starch, 10.0; peptone, 5.0; yeast extract, 5.0, with NaCl, 50.0; pH 7.0. Overnight grown culture (OD ~1.0) was used as inoculum. Media optimization for amylase production was carried out by “one-at-a-time approach” wherein single parameter was changed at a time while keeping others at a constant level. In order to see the effects of various nutritional and physical factors on amylase production, medium (A) was used as basal. Various carbon, nitrogen sources, and metal ions were varied, one at time, in the media. One percent of the mother culture was seeded as inoculum into 50 mL medium contained in 250 mL Erlenmeyer flask. The incubation was carried out at 35°C and 200 rpm for 72 h. The growth was monitored by recording the absorbance of the culture at 660 nm against uninoculated culture as blank.
For checking the amylase activity, cells were harvested after 72 h by centrifugation at 10,000 ×g for 10 min at 4°C. The cell-free supernatant filtered through a 0.22 μm cellulose acetate membrane (Millipore Corporation, MA, USA) was assayed. Effect of different parameters was monitored on the growth (A_660 nm) and amylase activity.