Wcx magnetic beads

All chemicals and solvents used were purchased from Sigma–Aldrich (St. Louis, MO). Solvents were HPLC grade and were used without further purification. Alpha-cyano-4-hydroxycinnamic acid (CHCA) was used as the MALDI matrix. WCX magnetic beads and Standard Preparation (Peptide Calibration Standard #206195 and Protein Calibration Standard I #206355) were purchased from Bruker Daltonik GmbH (Bremen, Germany).
The anti-α-tubulin antibody was from Epitomic Inc (Burlingame, California). Antibodies directed against 4E-BP1, phospho-4E-BP1 (Ser65), p70S6K1 and phospho-p70S6K1 (Thr389) were from Cell Signaling Technology, Inc (Danvers, USA). An IRDye 680-conjugated goat anti-rabbit IgG secondary antibody was from Rockland Immunochemical Inc (Gilbertsville, PA). The anti-eRF3b antibody directed against the human eRF3b was produced by Sangon (Shanghai, China).

We used MALDI-TOF-MS and magnetic beads to screen the serum samples to identify new biomarkers for colorectal cancer. The Sera were centrifuged at 10,000 g for 3 min at 4 °C after thawed on ice. Weak cation-exchange (WCX) magnetic beads (Bruker Daltonics, USA) were selected for the protein separation. According to the manufacturer’s protocol, first, 5 μL of serum was added to the mixture of 10 μL of beads slurry and 10 μL of binding solution in a 0.2-mL polypropylene tube. After incubation for 5 min, the supernatant was discarded. Then, the magnetic beads were washed with 100 μl wash buffer three times. Finally, the proteins were eluted from the magnetic beads with 5 μL elution buffer and mixed with 5 μL stable buffer. Finally, 1 μL protein mixture was deposited on a Micro SCOUT Plate (MSP). After natural drying, 1 μL CHCA matrix (3 mg/ml cyano-4-hydroxycinnamic acid, 50% ACN, 20% TFA) was added to each spot for detection.