How to prepare cell lysate for western blot?

PROTEIN EXTRACTION PROTOCOL:

1. Discard the medium in culture dishes with cells and wash the cells using ice-cold PBS.
2. Discard the PBS, add ice-cold lysis buffer.
3. Scrape the cells using cold plastic cell scraper. Collect the cells in microcentrifuge tubes.
4. Agitate the contents in microcentrifuge tubes for 30 min at 4 °C.
5. Centrifuge the tubes at 16,000 x g for 20 min at 4 °C. Collect the supernatant in fresh tube and place on ice. Discard the pellet.

NB: some specific proteins might need some specific treatment to be solubilized properly for extraction.

First, discard the cell culture medium, and wash the cells several times with 1×PBS, then solubilize the cells (approximately 2×10⁶-1×10⁷ cells) with 100 μL of 2×SDS-loading buffer. After this, boil the extracts at 100℃ for 5 min; and sonicate for 5-10 s, 3-4 times in total before loading.

The 2×SDS-Loading buffer includes 4% SDS, 20% glycerol, 200 mM DTT, 0.01% bromphenol blue and 0.1 M Tris-HCl, pH 6.8.