The activity of Xenopus oocytes and of bovine liver catalase (used as standard; SIGMA) was assayed spectrophotometrically, by following the disappearance of 30 mM H2O2 absorbance at 230 nm with an Perkin Elmer Lambda 5 recording spectrophotometer as was previously described52 (link). Briefly, 10–15 oocyte suspension in 1–2 ml of Ringer’s solution were sonicated (Bandelin Sonopuls sonifier, Bandelin Electronic, D12207 Berlin, Germany) at 50% power for 30 sec) and centrifuged at 10,000 × g at 4 C° for 5 min. Enzyme activity was assayed in the supernatant.
Sonopuls sonifier
Manufactured by Bandelin
Sourced in Germany
The Sonopuls Sonifier is a laboratory equipment designed for high-intensity ultrasonic cell disruption and homogenization. It utilizes ultrasonic energy to disrupt cells and break down larger particles into smaller ones.
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Lab products found in correlation
3 protocols using sonopuls sonifier
1
Enzymatic Activity Assay in Xenopus Oocytes
2
Yeast Cell Lysis and Protein Extraction
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After the treatment with H2O2 for 1 h at 30 °C/160 rpm, a centrifugation at 6000g for 5 min at 4 °C was done in order to harvest the yeast cells which were then washed three times with 20 mM Tris-HCl buffer (pH 7.5) and put back into suspension in the lysis buffer that contains 50 mM Tris-HCl buffer (pH 7.5), 1 mM ethylenediamine tetraacetic acid (EDTA), 10 mM 2-β- mercaptoethanol, 1 mM phenylmethylsulfony fluoride (PMSF), and 1% (v/v) glycerol at a ratio of 3 mL/g (wet weight). While disturbing the cells with cold, A Bandelin Sonopuls Sonifier (90%, 20 s, 12 × ) was then used, followed by a centrifugation (15,000g, 45 min at 4 °C) using a Sigma 2–16 K refrigerated centrifuge. The obtained supernatant was later on used for all enzyme activity assays. According to the Bradford procedure, the protein content was determined using bovine serum albumin (BSA) as standard (Bradford, 1976 (link)).
3
Tau Protein Oligomerization Assay
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In vitro oligomerization of recombinant truncated tau protein (aa 151–391, 100 μM) was carried out using heparin (Sigma-Aldrich, St. Louis, Missouri, United States) as an inducer at a final concentration of 25 μM in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) The reaction was performed overnight (for at least 12 hours) at 37°C. After incubation, tau oligomers were collected by centrifugation at 100,000 × g for 1 hour at room temperature and the pellet was re-suspended in PBS and sonicated for 5 seconds at 20% power output using an MS72 probe of a Bandelin Sonopuls Sonifier (Bandelin, Berlin, Germany). Subsequently, 1 μM aliquots were stored at -70°C. The oligomerization of the tau protein was verified by SDS gel electrophoresis, quantitative thioflavin T (ThT) fluorescence spectroscopy with excitation at 450 nm and emission at 510 nm, and by electron microscopy.
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