4, containing DNAse (10 μg mL−1), 4 mM phenylmethylsulfonyl fluoride and 5 mg of lysozyme and incubated for 30 min at 37 °C. Cells were disrupted on ice by sonification (35 W power for 10 min with 0.5-s pulses). Unbroken cell and cell debris were removed by centrifugation for 45 min at 10 000 g at 4 °C, and the supernatant was used as the crude cell extract. Membrane fractions were prepared by ultracentrifugation at 145 000 g for 2 h at 4 °C. The membrane pellet was resuspended directly in 50 mM 3-(N-morpholino)propane sulfonate, pH 7.0, buffer. The protein concentration of the subcellular fractions was determined (Lowry
et al., 1951) with bovine serum albumin as the standard. For overproduction of FocAStrep–N, cultures of E. coli BL21 containing pASK-IBA5focA were grown aerobically at 37 °C in 4 L of TB medium supplemented with Antidiabetic Compound Library solubility dmso 100 μg ampicillinmL−1 to an OD600 nm of approximately 0.5. Gene expression was induced by addition of 0.2 μg mL−1
anhydrotetracycline and cultures were incubated at 16 °C with continuous shaking for 14 h. Cells were harvested by centrifugation at 8000 g for 25 min and 4 °C. Cell Afatinib pellets were resuspended in 15 mL of buffer (50 mM Tris/HCl, 170 mM NaCl, pH 8.0) and were disrupted by sonification (35 W power for 10 min with 0.5-s pulses). The cell lysate was centrifuged for 30 min at 19 000 g at 4 °C. The resulting crude extract was again
centrifuged for 60 min at 100 000 g at 4 °C and the membrane fraction was resuspended in 5 mL buffer W (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0). The protein concentration was determined and 3 mg of n-dodecyl-β-maltoside (DDM) (Glycon Biochemicals, Luckenwalde, Germany) was added per milligram of protein under stirring and incubated at 4 °C overnight. The solution was centrifuged for 60 min at 100 000 g and 4 °C. The resulting supernatant containing solubilized Strep-tagged FocA was loaded onto a 5-mL column containing a Strep-Tactin-Sepharose (IBA, Göttingen) matrix. Further purification steps were carried out exactly as described in the IBA standard protocol under aerobic conditions at 4 °C (Schmidt & Skerra, 2007). The yield of FocAStrep–N was generally 1 mg L−1 of culture. Aliquots of 50-μg protein Selleck Ixazomib from crude extracts were separated on 10% w/v sodium dodecyl sulfate (SDS)-PAGE (Laemmli, 1970) and transferred to nitrocellulose membranes as described (Towbin et al., 1979.). Polyclonal antibodies raised against a FocA peptide (amino acids 141–159; Seqlab, Göttingen, Germany) were affinity purified after coupling of purified FocA to AminoLink® coupling gel (Pierce, Rockford, IL) exactly as described by the manufacturer. Elution of the bound antibodies was achieved using 50 mM glycine-HCl, pH 2.5, 0.1% w/v Triton X-100 and 0.15 M NaCl.
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