Methylomirabilis oxyfera whole-cell extracts were separated (30 μg of protein per lane) on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (Protran®, Germany) with a semi-dry transfer cell blotting system (Bio-Rad).
Blotting was performed click here at 100 mA for 45 min with a transfer buffer containing 25 mM Tris, 192 mM glycine and 20% methanol. After blotting, the blots were air-dried and stored at 4 °C until further use. For immunoblotting, the stored blots were washed in distilled water for 30 min. Subsequently, the blots were (1) incubated in blocking buffer (1% BSA) in Tris-buffered saline (TBS; 10 mM Tris-HCL, 0.9% NaCl, pH 7.4) for 1 h; (2) incubated for 1 h in either blocking buffer or rabbit preimmune buy Belnacasan serum diluted 250-fold in blocking buffer (negative controls) or antiserum diluted 250-fold in blocking buffer; (3) washed tree times for 10 min in TBS containing 0.05% Tween20; (4) incubated for 1 h in monoclonal mouse α-rabbit IgG alkaline phosphatase conjugate (γ-chain specific;
Sigma, The Netherlands) diluted 1500-fold in blocking buffer; (5) washed two times for 10 min in TBS containing 0.05% Tween and three times for 10 min in TBS; and (6) incubated for 5 min in alkaline phosphatase substrate BCIP/NBT (Sigma), rinsed in distilled water and air-dried. Cells from the M. oxyfera enrichment culture were chemically fixed by immersion in 2% paraformaldehyde and 0.2% glutaraldehyde
in 0.1 M PHEM buffer (25 mM HEPES, 10 mM EGTA, 60 mM PIPES, 2 mM MgCl2, pH 6.9) for 1 h, at room temperature, followed by overnight fixation at 4 °C. Next, the samples were washed three times with 0.1 M PHEM buffer pH 6.9 and resuspended in 12% gelatin in 0.1 M PHEM buffer pH 6.9 at 37 °C. After 5 min at 37 °C, the samples were pelleted by a centrifugation step, half of the supernatant was removed, and the samples were placed on ice for 15 min. The gelatin-embedded cells were selleck inhibitor cut into small cubes (1–2 mm3) under a stereo microscope, infiltrated overnight in rotating vials at 4 °C with 2.3 M sucrose in 0.1 M PHEM buffer pH 6.9 and frozen in liquid nitrogen. Cryosectioning was performed in a cryoultramicrotome (UCT/FCS or UC6/FCS; Leica Microsystems, Vienna, Austria). Ultrathin cryosections (about 70-nm) were picked up with a drop of 1% methylcellulose and 1.15 M sucrose in PHEM buffer and transferred to carbon-formvar-coated grids (copper, hexagonal 100-mesh) for immunogold localization. For single labelling, grids containing ultrathin sections of M.
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