Whole cell lysates (WCLs) of eukaryotic cells
| Materials | |
|---|---|
| PBS 1X | |
| RIPA buffer | |
| Sepharose Beads |
Procedure
All following steps are performed in the cold room:
- Aspirate cell culture medium with a pasteur pipette
- Rince attaching cells carefully with cold PBS
- Add 1 mL RIPA buffer per 10 cm dish or 0.5 mL per 6 cm dish and incubate for approx. 2 min at 4°C
- Carefully detach the cells with a cell scraper and transfer the lysate in a fresh tube
- Mechanical shear the DNA by passing it multiple times through a fine needle
- Add 50 μL sepharose beads to the lysate
- Mix the suspension by overhead rotation for 5 min at 4°C
- Centrifuge lysate for 30min at 13.000 rpm, 4°C
- Transfer 100 μL of the supernatant into fresh tubes and mix 1:2 with SDS buffer, boil the sample briefly
- Store the remaining lysate at -80°C or mix 1:3 with RIPA buffer for the direct usage in a GST-Pulldown experiment