, Sweden; purified E. coli AP, DNP, CCCP, antibody to GroEL, 4-chloro-1-napthol and Freunds adjuvant from Sigma-Aldrich, USA; Ni-NTA Agarose from QIAGEN, Germany; HRP-conjugated goat anti-rabbit IgG (secondary antibody) and proteinA-CL agarose from Genei, India; the Nitrocellulose transfer membrane from BioRad Laboratories, USA; 35S-methionine from Board of Radiation and Isotope Technology, India; H2O2, Tween-20 and anti-DnaK antibody from Merck, India; Isopropyl β-D-thiogalacto pyranoside (IPTG) and p-nitrophenyl phosphate (PNPP) from Sisco Research Laboratories, India. Western blot experiment This experiment was performed according to the method described in [13]. Interested

specific protein on the blotted membrane was identified by using the antiserum of the protein (raised selleck kinase inhibitor in rabbit) as the primary antibody, HRP-conjugated goat anti-rabbit IgG as the secondary antibody and 4-chloro-1-napthol and H2O2 as the HRP substrates. Pulse-label/Pulse-chase and immunoprecipitation experiments Cells of E. coli Mph42 were initially grown to the log phase (up to [OD]600 nm ≈ 0.3, i.e., 1.5 × 108 cells/ml) at 30°C in MOPS www.selleckchem.com/products/KU-55933.html medium (where the methionine concentration was 1/10th of the normal MOPS medium [18]) and were Epigenetics inhibitor subsequently transferred to the methionine-free MOPS medium. For pulse-label and immunoprecipitation experiment, log phase grown cells in methionine-free MOPS medium were allowed

to grow further at 30°C. At different instants of growth, 1 ml cell aliquot was withdrawn to label with 35S-methionine (100 μCi/ml) for 1 min. The labeled cells were

treated with 5% Trichloroacitic acid. The protein precipitate was washed with 80% cold acetone. The air dried precipitate was suspended in 50 μl of 50 mM Tris buffer (pH 8.0) containing 1% SDS and 1 mM EDTA. It was then heated at 100°C for 3 min; 30 μl of this sample was diluted with 1 ml of Triton X-100 buffer [2% Triton X-100, 50 mM Tris, pH 8.0, 150 mM NaCl and 1 mM EDTA] and centrifuged to remove nonspecific precipitates. From the supernatant, for immunoprecipitation of any protein, requisite amount of the antibody to that protein was added and subsequently incubated overnight at 0°C. To this incubated Cobimetinib research buy sample, 50 μl of proteinA-CL agarose was added and incubated further at 0°C for 20 min. The immunocomplex was washed and finally suspended in 50 μl of 2× sample buffer [19], heated at 100°C for 3 min prior to loading on 12% SDS-polyacrylamide gel for electrophoresis; finally phosphorimaging of the gel was performed in Typhoon 9210 (GE Health Care). For pulse-chase and immunoprecipitation experiment, log phase grown cells in methionine-free MOPS medium were radio-labeled with 35S-methionine (at a concentration of 30 μCi/ml of cell culture) for the required time and the label was subsequently chased by 0.2 M cold methionine. At different instants of chasing, cell aliquot was withdrawn to extract proteins by the method of Oliver and Beckwith [19].