by Proteomics 5: 2331C2339 [PubMed] [Google Scholar] 33. abundance percentage and subcellular localization, and suggested a complete surface picture of the cell by integrating all the obtained information. METHODS and MATERIALS Strains, tradition circumstances, and antibodies. stress 163K (ATCC 43663) was expanded at 25C Bakuchiol in Aluotto moderate (3, 24). Cells had been cultured to attain an optimal denseness at 600 nm of 0.07. Monoclonal antibodies had been raised against the complete cell surface area of for 10 min at 4C and washed 3 x with ice-chilled PBS-1 moderate comprising 75 mM sodium phosphate (pH 7.3) and 68 mM NaCl, suspended in a 10-ml solution of Sulfo-NHS-LC-Biotin in PBS-1, and kept for 30 min on ice. To quench the reaction and remove the excess biotin reagent, the cells were washed three times with 0.1 mM glycine (pH 6.1). Identification of surface proteins. The biotinylated surface proteins were isolated and identified, referring to the procedure of a previous study (33). The cell membrane fraction was isolated through osmotic lysis as described previously (30), with slight modifications. Cells from 1 liter of culture were labeled as described above when necessary and suspended in 1 ml of 4 M glycerol containing 0.5 mM phenylmethylsulfonyl fluoride, mixed with 1 ml of 5 M NaCl, and incubated at 37C for 10 min. The cell suspension was dispersed rapidly from a syringe into 100 ml of water kept at 37C and then incubated for 15 min. The cell membranes were collected by centrifugation at 34,000 for 30 min and washed with PBS-2 medium consisting of 100 mM sodium phosphate and 150 mM NaCl (pH 7.0). The cell membranes suspended in 10 ml of PBS-2 Bakuchiol were solubilized with 2% Zwittergent 3-14 for 1 h at 4C with head-over-head mixing. The insoluble fraction was removed by centrifugation at 100,000 and 4C for 1 h. The soluble fraction was diluted to a 1-mg/ml protein concentration using PBS-2 and subjected to 1 ml of avidin-agarose matrix (Thermo Scientific) equilibrated in PBS-2. The matrix was washed 10 times with 1 ml of PBS-2, and the trapped proteins were eluted using glycine-HCl (pH 2.7) and concentrated using Centrifugal Ultracel-3K filters (Millipore, Bedford, MA). Separately, the hydrophobic proteins were collected by Triton X-114 phase partitioning as described previously (4). To detect biotin labeling, the proteins developed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were transferred onto a nylon sheet, treated by streptavidin conjugated with horseradish peroxidase (HRP; GE Healthcare, Milwaukee, WI), and visualized by chemiluminescence. For peptide mass fingerprinting (PMF), the protein bands or spots were excised manually and treated as described previously (25, 26, 35). Quantification of surface protein. Two-dimensional gel electrophoresis was performed as described previously (29). Briefly, the cell membrane was lysed by a lysis buffer. The insoluble fraction was removed by centrifugation at 100,000 and 4C for 30 min, and a 20-l soluble fraction containing 30 g of protein was subjected to an isoelectric gel and then to Bakuchiol SDS-PAGE. To quantify the protein amounts, the gel was stained by Coomassie brilliant blue (CBB) staining, scanned by a transparent scanner (GT9800F; Epson, Nagano, Japan), and analyzed by ImageJ 1.37v (http://rsb.info.nih.gov/ij/) (2). Microscopy. Mycoplasma cells were bound to glass, fixed chemically, stained, and observed as previously described (19, 34, 39, 42) using 3.3 pM antibodies or 10 g of streptavidin/ml conjugated with Cy3 (GE Healthcare). The cell fractions were observed as they were for the fixed cells on glass slides. Sequence analysis. The transmembrane segment, signal peptide, hydropathicity, and lipoprotein were predicted by SMART 6 (http://smart.embl-heidelberg.de/), SignalP 3.0 Server (http://www.cbs.dtu.dk/service/SignalP/), ProtParam (http://web.expasy.org/protparam/), and DOLOP (http://www.mrc-lmb.cam.ac.uk/genomes/dolop/), respectively. RESULTS Labeling cell surface proteins. To identify cell surface proteins systematically, we labeled the surface proteins by using Sulfo-NHS-LC-Biotin, a hydrophobic biotinylation reagent (33). Whole cells suspended in PBS were treated with various concentrations of Sulfo-NHS-LC-Biotin. After cell lysis, proteins were separated by SDS-PAGE and blotted onto a nylon sheet, and biotinylated proteins were detected using streptavidin-HRP (Fig. 1A). The proteins were biotinylated according to the concentration of Sulfo-NHS-LC-Biotin used. The relative intensities of signals among Bakuchiol proteins did not KNTC2 antibody agree with the CBB-stained band intensities, suggesting that the proteins exposed on the cell surface (surface proteins) were labeled preferentially. To visualize the biotinylation of surface proteins, the labeled cells were bound to glass, fixed chemically, and.
