• Cell Extraction Buffer
• Phosphate buffered saline
• Pipette and pipette tips
• 15 ml conical tube
• 1.5 ml Eppendorf tubes
1. Media was removed from cells using pipette and suspended cells were transferred to a 15 ml conical tube.
2. Cells adhered to plate were washed twice with 3 ml phosphate buffered saline, then the solution was aspirated.
3. One millilitre of trypsin-EDTA was added to plate to remove adhered cells. After 2-3 minutes the adhered cells lifted off the bottom of the plate.
4. Detached cells were added to the same 15 ml conical tube as the suspended ones.
5. Steps 1-4 were repeated for every treatment plate.
6. The 15 ml conical tubes were centrifuged for 5 minutes at 1000 rpm and the media supernatants were aspirated from the cell plates.
7. Phosphate buffered saline was aspirated from the top and 1 ml of Cell Extraction Buffer was added to each pellet to lyse cells.
8. Cells were left on ice for 30 minutes and vortexed at a setting of 3 (any higher would cause denaturing of protein) every 10 minutes.
9. Cell lysates were transferred to 1.5 ml Eppendorf tubes and centrifuged at 13 000 rpm for 10 minutes at 4°C.
10. Clear lysate was transferred to clean 1.5 ml Eppendorf tubes.
Pictures from Procedure (click on each picture to open it in its own window)
Interior of Cell Culture Room
Interior of Centrifuge
Labelled Cell Plates