The GCF protein extracts were subjected to SDS-PAGE analysis under reducing conditions. An aliquot of each pool (8 µg of protein content) was combined with 2× Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue) including 0.5% 2-mercaptoethanol. Protein denaturation was obtained by heating the mixture at +95 °C for 5 min in a Thermomixer Comfort device (Eppendorf, Milan, Italy). Protein separation was then carried out using precast gel BoltTM 12% Bis-Tris Plus and 4–12% Bis-Tris Plus, 12 wells. The electrophoretic run was performed in a Mini Protean Tetra Cell device (Bio-Rad, Hercules, CA, USA) using MES SDS 1× as the running buffer, at 100 V for 30 min, and next at 200 V for the remaining run time. After rinsing with pure water, the gels were stained overnight at constant moderate shaking with Coomassie Blue G-250, and de-stained with 30% methanol/10% glacial acetic acid solution. The GS-800 Calibrated Imaging Densitometer (Bio-Rad, Hercules, CA, USA) was used to acquire the gel images.
Thermomixer comfort device
Manufactured by Eppendorf
Sourced in Italy, Germany
The Thermomixer Comfort device is a high-performance mixing and temperature control instrument designed for a wide range of applications in life science laboratories. It provides precise temperature regulation and efficient mixing for sample preparation, incubation, and reaction processes.
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Lab products found in correlation
2 protocols using thermomixer comfort device
1
SDS-PAGE Analysis of GCF Protein Extracts
2
Protein Precipitation and Solubilization
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Proteins in the collected fluid were precipitated immediately by adding five volumes of cold 0.1 M ammonium acetate in methanol, containing 0.07% β-mercaptoethanol. Samples were kept overnight at -20ºC and then centrifuged at 20,000 xg for 20 min. The pellet was washed twice with cold methanol, dried with N 2 gas and solubilized in a sample rehydration buffer containing 8 M urea, 2% (w/v) CHAPS, 50 mM DTT, 2 mM PMSF and 0.2% (v/v) IPG buffer pH 3-10 (GE Healthcare, Uppsala, Sweden). After rehydration, samples were incubated in a Thermomixer comfort device (Eppendorf AG, Hamburg, Germany) at 42 ºC and 1,000 rpm during 3 h, then centrifuged at 10,000 xg for 10 min at RT and filtered (0.45 µm ultrafree-MC filters, Millipore, Bedford, USA). Protein concentration in the samples was quantified immediately with the Bradford method using an Asys UVM 340 spectrophotometer with microtiter plates (Biochrom Ltd., Cambridge, UK) and BSA as standard.
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