Western-blotting (Foxo1)
- Culture cells (MEFs etc) in plate with 10 cm in
diameter, 37C, 5% CO2 incubator.
- Starve the cells overnight for at least 18 hours with
medium containing no serum.
- Treat the cells with insulin (100 nm) or others for a
time period of 30 min.
- Wash the cells with cold PBS twice.
- Add the cell lysis buffer (900 ul).
- Place the cells in 4C cold room and shake for 30 min.
- Harvest the cells into 1.5 ml eppendof tubes.
- Centrifuge the tubes at 14,000g for 10 min at 4 C.
- Aspirate the supernatant for storage at –80C.
Otherwise continue experiments followed.
- Take 5 ul of the cell lysates for protein assay by Bio
Protein Assay Reagent.
- 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.
- Transfer the protein from the gel to NC membrane in
Tobin buffer with 20 % methanol at 110V for 1 hour.
- Block the membrane with 3% milk in Washing buffer for
1 hour at RT.
- Incubate the membrane with the primary antibody in
Blocking milk overnight at 4C.
- Wash the membrane three times with Washing buffer, 15
min each time.
- Incubate the membrane with secondary antibody in
Blocking milk at RT for 1 hour.
- Wash the membrane three times with Washing buffer, 15
min each time.
- Add 2 ml of ECL solution (1:1) to each membrane for 1
min.
- Expose the film for 25 seconds or more and develop the
film.