If not stated otherwise, chemicals were obtained from Merck (Darmstadt, Germany) or Carl Roth GmbH (Karlsruhe, Germany); columns for protein purification were obtained from Cytiva (formerly GE Healthcare Life Sciences (Uppsala, Sweden)). [9,10-3H(N)]-triolein was obtained from PerkinElmer Life Sciences (Waltham, MA, USA). Pierce™ Unstained Protein MW Marker from Thermo Fisher Scientific™ was used as size marker for SDS-PAGE gels. Blue Prestained protein standard broad range marker from New England BioLabs was used for SDS-PAGE and for immuno blotting. Disruption of cells was carried out using a homogenizer (SONOPLUS ultrasonic homogenizer HD 2070, Bandelin, Berlin, Germany).
Sonoplus ultrasonic homogenizer hd 2070
Manufactured by Bandelin
Sourced in Germany
The SONOPLUS ultrasonic homogenizer HD 2070 is a laboratory equipment designed for mechanical disruption and homogenization of liquid samples. It operates using high-frequency ultrasonic waves to break down particles and mix components in a sample.
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4 protocols using sonoplus ultrasonic homogenizer hd 2070
1
Protein Purification and Analysis
2
Purification of Recombinant Murine Proteins
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Human his6-smt-G0S2 constructs were transformed into Escherichia coli BL21(DE3) CodonPlus® cells (Stratagene, La Jolla, CA). Cultures were grown at 37 °C on selective LB medium containing 40 μg/ml kanamycin to an A600 of 0.5. Expression was induced by the addition of 0.5 mm isopropyl-β-d -thiogalactopyranoside at 30 °C. After 15 h of induction, cells were harvested, resuspended in sucrose solution (250 mm sucrose, 1 mm EDTA, 1 mm DTT, 20 μg/ml leupeptin, 2 μg/ml antipain, 1 μg/ml pepstatin, pH 7.0), and disrupted by sonication (SONOPLUS ultrasonic homogenizer HD 2070, Bandelin, Berlin, Germany) on ice. After centrifugation at 15,000 × g for 20 min at 4 °C, the supernatants were collected. Protein concentrations were determined as described below. Expression of the murine ATGL-Strep fusion (Strep-mATGL) and His6-Smt-mCGI-58 in E. coli is described in Refs. 13 (link) and 42 (link), respectively.
3
Catalase Activity Assay in Fibroblasts
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For catalase (EC 1.11.1.6.; 2H2O2 oxidoreductase) activity assay, fibroblasts were rinsed three times with 0.2 M PBS pH 7.4, rubber scraped, centrifuged at 450 g for 7–10 min at 4°C, and suspended in 1 mL of cold buffer (50 mM potassium phosphate pH 7, containing 1 mM EDTA). 3–5 × 106 fibroblasts were sonicated for four cycles on ice (40% of amplitude, 10 s of sonication, and 5 s of pause) (Sonoplus Ultrasonic homogenizer HD 2070, Bandelin electronic, Berlin, Germany) and then centrifuged at 10000 g for 15 min at 4°C. Catalase activity was determined in the supernatants by using the FR20 kit following the manufacturer's instructions (Oxford Biomedical research, Oxford, USA).
4
Lipid Peroxidation Measurement Protocol
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Polyunsaturated fatty acids generate malondialdehyde (MDA) upon oxidative decomposition. Thus, the adduct 1:2 that one molecule of MDA forms with two molecules of TBA is considered as an indirect measurement of plasma membrane lipid peroxidation [37 (link)]. PBLs (15 × 106) were rinsed three times with filtered PBS (0.2 M), pH 7.4, and sonicated for four cycles on ice by setting for each cycle 40% of amplitude, 10 s of sonication and 5 s of pause (Sonoplus Ultrasonic homogenizer HD 2070; Bandelin Electronic, Berlin, Germany). Proteins were precipitated by adding cold 10% (w/v in water) trichloroacetic acid, then cell lysate was incubated with 16-mM TBA (prepared in 10-μM NaOH) in a water bath at 90 °C for 45 min. Reaction was stopped by placing the reaction mix on ice for 5 min. After another 5 min at room temperature (RT), the 1:2 adduct was extracted by using 500 μL of n-butanol and 50 μL of a saturated solution of NaCl (JB Baker, Deventer, Holland). Samples were centrifuged at 12 × 103 rpm for 1 min., then the absorbance of the supernatant was read by using an Ultrospec 4000 UV/visible spectrophotometer (Pharmacia Biotech, Stockholm, Sweden) set at 532 nm. A positive control of reaction 0.5-mM MDA was used instead of cell lysate.
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