Pierce protease inhibitor tablet

The cell pellet was resuspended in the lysis buffer [50 mM Tris (pH 8.0), 100 mM NaCl, 10 mM imidazole (pH 8.0) 10 mM beta-mercaptoethanol (BME), Pierce^TM protease inhibitor tablet (catalog # A32963; Thermo Scientific)] and stored at −80 °C. To purify the protein, the frozen pellet was thawed in the presence of 0.25 mg/mL lysozyme. The cells were disrupted using a sonicator (ON/OFF/total-time:0.5/0.5/120 s). The cell lysate was centrifuged at 25,000 g for 20 min and the supernatant was loaded on nickel rapid run agarose beads (Goldbio) that were previously equilibrated with the wash buffer [50 mM Tris (pH 8.0), 100 mM NaCl, 10 mM imidazole (pH 8.0), 5 mM BME and Pierce^TM protease inhibitor tablet (catalog # A32963; Thermo Scientific). The beads were washed three times with five-column volume of wash buffer and finally eluted with 3-column volume of the elution buffer [50 mM Tris (pH 8.0), 100 mM NaCl, 500 mM imidazole (pH 8.0)]. The eluted protein sample was further purified by gel purification using HiLoad 16/600 Superdex 200 pg column (GE Healthcare) equilibrated with 10 mM Hepes (pH 7.4), 100 mM NaCl, 5 mM DTT. The fractions containing the protein of interest were pooled and concentrated using a 10 K MWCO Amicon centrifugal filter (Millipore). During all steps, the sample was kept at 4 °C.

For protein expression and purification, pET3a plasmids encoding c-terminal histidine-tagged ColN was provided by Professor Jeremy H. Lakey, Newcastle University, UK. Luria-Bertani broth and agar were bought from Hardy Diagnostics (Santa Maria, CA, USA). L-(+)-arabinose was obtained from TCI (Tokyo, Japan). Imidazole, DNase I, and ampicillin sodium salt were purchased from PanReac Applichem (Darmstadt, DE, USA). PierceTM protease inhibitor tablets and bicinchoninic acid (BCA) protein assay kit was purchased from Thermo Scientific (Waltham, MA, USA). 10X Phosphate Buffered Saline (PBS), skim milk powder and sodium chloride (NaCl) were purchased from Vivantis Technologies (Selangor, MY) and Ajax Finechem (Seven Hills, NSW 2147, Australia).
For cell culture, DMEM/Ham’s F-12, DMEM high glucose and 1X HEPES were bought from HyClone (Logan, Utah), 10% fetal bovine serum (FBS) from Merck (Darmstadt, Germany), 1% penicilin/streptomycin from Gibco (Gaithersburg, MA, USA). The reagents 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Abcam (Cambridge, CB2 0AX, UK). Cisplatin and Doxorubicin were bought from Sigma Chemical, Inc. (St. Louis, MO, USA).

Fetal bovine serum (FBS), penicillin, streptomycin, trypsin, tryptan blue, Bradford’s reagent, phosphate-buffered saline (PBS), cisplatin, and paclitaxel were acquired from Sigma-Aldrich (Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from PAN Biotech (Aidenbach, Germany). Acetonitrile (ACN), deionized water, LC-MS CHROMASOLV, and methanol (≥99.9%) were purchased from Honeywell (Charlotte, USA). Formic acid (FA) and trifluoroacetic acid (TFA) were procured from Fisher Scientific (Loughborough, UK), whereas hydrochloric acid (HCl, 37%) was sourced from VWR Chemicals (Paris, France). C18 STAGE (stop and go extraction) tips, lysis buffer, lysyl-endopeptidase (Lys-C), and Pierce^TM protease inhibitor tablets were purchased from Thermo Fisher Scientific (Rockford, USA).