Cell harvest for Western Blot
- Aspirate media from dish and scrape cells. Resuspend cells in 5ml of serum-free media or PBS and gently spin down cells.
- 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.
- Transfer to an epi tube and spin at 13K for 15min at 4°C to pellet debris.
- 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:
- Boil samples (which have dye in them) for 5 min and then place IMMEDIATELY on ice.
- Quick spin the samples to collect condensation. Load gel and run.
