Anti protease inhibitor cocktail complete

Solutions were prepared with Milli-Q water. Water of HPLC grade, trifluoroacetic acid (TFA), isopropyl alcohol, tributyl phosphate (TBP), ammonium bicarbonate (ABC), dithiothreitol (DTT), iodoacetamide (IAA), urea, Triton X-100, Tween-20, Trizma-base, MES, sucrose, BCA protein assay reagent and BSA were from SIGMA Chemical Co. (St. Louis, MO, USA). Acetonitrile (ACN), methanol, and glycerol were from Merck (Darmstadt, Germany). Porcine trypsin was from Promega (Madison, WI, USA). Bradford proteinbinding colorimetric assay was purchased from Bio-Rad (Hercules, CA, USA). Anti-protease inhibitor cocktail (Complete) was from Roche (Monza, Italy). NuPAGE s SDS-PAGE gel electrophoresis system components (mini gels, running and loading buffers, molecular weight markers and Coomassie brilliant blue staining-CBB) were supplied by Life Technologies (Paisley, Renfrewshire, UK). Hybond-ECL nitrocellulose membrane was from GE (Little Chalfont, Buckinghamshire, UK). Anti-aminopeptidase N (CD13) monoclonal antibody, anti-annexin A2 (ANXA2) and anti-carbonic anhydrase (CA2) polyclonal antibodies were from Abcam (Cambridge, UK). Species-specific secondary peroxidase conjugated antibodies and ECL reagents were from Pierce (Rockford, IL, USA).