Six microliters of each sample was injected and peptides were purified and concentrated on a C18-PepMap precolumn (0.3 mm ID 65 mm, 100? pore size, 3 mm particle size, Dionex) at a flow rate of 0.02 mL/min. complex disease with predisposing genetic variants and environmental factors that contribute to the illness as a whole. In recent Cyclosporin A years, many studies reported that the incidence of colorectal cancer is increased by 4% per year in China[1C3]. Hb3 is an anti-colorectal cancer monoclonal antibody produced in our laboratory. Its sensitivity and specificity are superior to those of anti-CEA. Monoclonal antibody Hb3 (mAb Hb3) targets the Hb3 antigen which is expressed in 85% of colorectal cancers, showing that CA-Hb3 may be useful in the diagnosis of colorectal cancer. In this study, using SDS-polyacrylamide gel electrophoresis (PAGE) and microcapillary reversed-phase liquid chromatography-tandem mass spectrometry, we have identified 5 proteins, including one membrane protein which was initially determined to be Ca-Hb3 by bioinformatics analysis. MATERIALS AND METHODS Preparation of mAb Hb3 mAb Hb3, a murine IgM-type mAb, was prepared using 5 108 Hb3 hybridoma cells (our laboratory) to immunize Balb/c mice. Mice were sacrificed and ascites was extracted to obtain supernatant by centrifugation. Instrumentation NanoLC-MS was performed online with a Waters capillary LC system interfaced with a Waters (Micromass) electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer. Cells and cell Cyclosporin A culture Human colon carcinoma HRT-18 cells were cultured in RPMI 1640 medium (Promega Inc.) supplemented with 100 mL/L heat-inactivated fetal bovine serum (FBS, Hangzhou Sijiqing BRL Inc.), 30 g/L em L /em -glutamine, 100 kg/L streptomycin and 100 U/L penicillin at 37C in a humidified atmosphere containing 50 mL/L CO2. SDS-PAGE and Western blot analysis Total cell extraction from human colon carcinoma HRT-18 cells was performed by Western blot assay. Samples in the cold lysate buffer (50 mmol/L Tris-Cl pH 8.0, 150 mmol/L NaCl, 10 mmol/L Triton X-100, 10 mmol/L PMSF) were electrophoresed on 10% gradient SDS-polyacrylamide gels in the presence of -mercaptoethanol. Half of the proteins were transblotted onto a NC membrane, which was blocked with 50 mg/mL fat-free milk and 100 mL/TBS for 2 h and incubated with primary antibody (mAb Hb3) overnight at 4C. The membrane was washed and incubated with HRP-conjugated goat anti-mouse IgG/IgM (SIGMA Inc.) at room temperature for 1 h. The membrane was washed Cyclosporin A and the antigen-antibody reaction was visualized using an ECL detection system (KPL Inc.). The other half of proteins were stained with Coomassie blue. Trypsin digestion Bands of interest were excised and minced into 1 mm3 pieces with sterile razor blade. The pieces were washed 3 times with ddH2O and then with 50% acetonitrile in 100 mmol/L NH4HCO3, incubated in water bath for 60 min if necessary. The liquid was discarded, 100 L 1% acetonitrile was added and the dried acetonitrile was removed after 10 min. For SDS-PAGE gel bands, gel pieces were rehydrated Rabbit Polyclonal to RNF111 and proteins were reduced with 200 L of 100 mmol/L DTT in 100 mmol/L ammonium bicarbonate for 30 min at 56C. The reduction solution was discarded and 100 L of 50 mmol/L iodoacetamide in 100 mmol/L ammonium bicarbonate was added. Incubation lasted for 30 min at room temperature in the dark. Gel bits were washed with 500 L of 25 Cyclosporin A mmol/L ammonium bicarbonate in 500 mL/L acetonitrile for 15 min, then 100 L 100% acetonitrile was added and the dried acetonitrile was removed after 10 min. The gel pieces were rehydrated by adding 5 L of trypsin in 40 mmol/L ammonium bicarbonate and incubated overnight at 37C. The supernatant was removed and placed into a new tube. Peptides were extracted from the gel pieces with 50 L of 20 mmol/L ammonium bicarbonate for 20 min and then with 50 L of 5% formic acid in 50% acetonitrile for 2 20 min. The extracts were added to the supernatant. The mixed solution was dried using a Speed-Vas. Nano-HPLC tandem.