Cell harvest for Western Blot


  1.  Aspirate media from dish and scrape cells.  Resuspend cells in 5ml of serum-free media or PBS and gently spin down cells.
  2. Aspirate supernatant and then add lysis buffer to pellet (200µl for 10cm plate or 500µl for a 15cm plate).  Resuspend and leave on ice or 4°C for 30min to lyse.
  3. Transfer to an epi tube and spin at 13K for 15min at 4°C to pellet debris.
  4. At this point you can freeze the protein lysates at –20°C until use
    Measure protein concentration using Lowery or Bradford assays
    For Western- you will run 50µg of protein
    To do this: Dilute protein if too concentrated to a volume ~20µl
    Add loading dye buffer (6X) to get a 1X final concentration.
    (For example add 5µl of dye buffer to 30µl of lysate…)
    At this point if you want, you can store the samples at –20°C until you run the gel.

Before loading the gel, do the following to the samples:

  1. Boil samples (which have dye in them) for 5 min and then place IMMEDIATELY on ice.
  2. Quick spin the samples to collect condensation. Load gel and run.