Western-blotting (Foxo1)

 

  1. Culture cells (MEFs etc) in plate with 10 cm in diameter, 37C, 5% CO2 incubator.
  2. Starve the cells overnight for at least 18 hours with medium containing no serum.
  3. Treat the cells with insulin (100 nm) or others for a time period of 30 min.
  4. Wash the cells with cold PBS twice.
  5. Add the cell lysis buffer (900 ul).
  6. Place the cells in 4C cold room and shake for 30 min.
  7. Harvest the cells into 1.5 ml eppendof tubes.
  8. Centrifuge the tubes at 14,000g for 10 min at 4 C.
  9. Aspirate the supernatant for storage at 80C. Otherwise continue experiments followed.
  10. Take 5 ul of the cell lysates for protein assay by Bio Protein Assay Reagent.
  11. Take 52.5 ul of the cell lysates (around 20 ug of protein) which is maximal volume (70 ul) for 10 well-mini-gel (15 mm thick) and add 17.5 ul of 4* SDS loading buffer. Heat the sample at 100C for 5 min and cool down at RT for 10 min before loading. Run the gel in SDS-Glycine running buffer at 50V for 40 min and then 110 V for 2 hours.
  12. Transfer the protein from the gel to NC membrane in Tobin buffer with 20 % methanol at 110V for 1 hour.
  13. Block the membrane with 3% milk in Washing buffer for 1 hour at RT.
  14. Incubate the membrane with the primary antibody in Blocking milk overnight at 4C.
  15. Wash the membrane three times with Washing buffer, 15 min each time.
  16. Incubate the membrane with secondary antibody in Blocking milk at RT for 1 hour.
  17. Wash the membrane three times with Washing buffer, 15 min each time.
  18. Add 2 ml of ECL solution (1:1) to each membrane for 1 min.
  19. Expose the film for 25 seconds or more and develop the film.