Cell Extraction of Protein
NOTE: Due to University Regulations, Tissue Culture and Cell Extraction were observed. When MPP+ was added to the cells, I was not present for safety reasons.
Materials
• Cell Extraction Buffer
• Phosphate buffered saline
• Pipette and pipette tips
• 15 ml conical tube
• Centrifuge
• Aspirator
• Vortex
• 1.5 ml Eppendorf tubesProcedure
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)
