Gel filtration standard 1511901

For characterization by SEC-MALS, purified mCes2c (1.5 mg/mL) was centrifuged at 12,851× g for 15 min at 4 °C prior the actual experiment. mCes2c was eluted from a Superdex 200 Increase 10/300 GL column (GE Healthcare) in a buffer B containing 20 mM potassium phosphate, pH 7.4 and 150 mM NaCl with a flow rate of 0.3 mL/min at 8 °C directly coupled to a MALS unit from Wyatt Technologies (TREOS II) equipped with three detection angles. Data were analyzed with the program ASTRA 7.1.2.5 (Wyatt Technologies). To determine the molecular weight based on the SEC, protein standard from Bio-Rad (Gel filtration Standard, 1511901) was used (equation for linear regression: y = −0.1963x + 4.6251; R² = 0.9984).

Cell pellets were resuspended in Extraction buffer (20 mM Tris-HCl pH 7.4; 10 mM MgAcetate; 40 mM NH₄Cl; 1m M DTT) and lysed in the presence of allumina (2 g/g pellet) and 0.01 mg DNase (Sigma-Aldrich, Saint Louis, MS, USA). Lysates were centrifuged at 26,000× g for 30 min at 4 °C. Cleared extracts (0.5 mg) were loaded onto 12 mL continuous 5–15% glycerol gradients in sterile Extraction buffer, with and without pre-incubation with 0.05 mg RNase A (Sigma-Aldrich) for 30 min at 37 °C, and then centrifuged at 36,000 rpm for 17 h at 4 °C (rotor model SW40Ti; Beckman Coulter, Brea, CA, USA); fractions of 0.5 mL were collected using an AKTA FPLC machine. Collected fractions were precipitated with trichloroacetic acid (final concentration 10%) for 1h. Samples were then centrifuged at 15,000× g for 30 min at 4 °C, washed with 0.5 mL of cold acetone and centrifuged again under the same conditions. Proteins were resuspended in SDS sample buffer (64 mM Tris-HCl pH 6.8, 15% 2-mercaptoethanol, 10% glycerol, 2% SDS, 0.1% bromophenol blue) and used for Western blotting analysis. The experiment was repeated using a molecular mass standard (Gel Filtration Standard #1511901, Biorad, Hercules, CA, USA).