This article details a protocol for extracting proteins from neuronal cancer cells cultured on coverslips. The method involves cell lysis under cold conditions to preserve protein integrity for subsequent analysis.
Take a cell culture containing neuronal cancer cells attached to a coverslip and maintain it under cold conditions.
Remove the culture medium and repeatedly wash the cells with cold phosphate buffer to eliminate the residual medium.
Add a lysis buffer containing a low concentration of non-ionic detergent and protease inhibitors.
The non-ionic detergent disrupts the cell membrane to initiate lysis.
Meanwhile, the protease inhibitors block the cellular proteolytic enzymes, protecting the proteins from enzymatic degradation.
Next, use a cell scraper to dislodge the partially lysed cells from the coverslip.
Transfer the contents to a tube containing the lysis buffer.
Under cold conditions, use a homogenizer to mechanically lyse the cells, ensuring complete cell lysis and protein release.
Centrifuge the cell lysate.
Collect the supernatant into a fresh tube.
The extracted proteins from the neuronal tumor cells are now ready for further analysis.
For protein expression validation, 48 hours after transfection, place the culture dishes on ice and wash the cells two times with PBS. Treat the cells in each dish with 200 to 400 microliters of lysis buffer, and use a cell scraper to remove the cells from the coverslips.
Transfer the detached cells from each plate into individual microcentrifuge tubes, and further dissociate the cells with a homogenizer under ice. After one minute, pellet the homogenized cell pieces by centrifugation.
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