During the sample preparation of cell lysate, add cell lysis buffer, agitate the cells, centrifuge, and recover and aliquot supernatant. What is the procedure/protocol for cell pellets preparation for western blotting? I am uncertain whether to store the cell pellets after centrifugation at -80 degree Celsius or add cell lysis buffer to cell pellets and store at -80 degree Celsius.
Thank you for your response.
Michael Essien Sakyi
Hi,
There is 2 option
Both options are commonly used and depend on your experimental needs. If you anticipate using the cell lysates for multiple experiments or want to store them for a longer time, option 1 (storing the cell pellets alone) is preferable. If you require immediate access to the lysates or prefer the convenience of pre-lysed samples, option 2 (adding lysis buffer before freezing) would be more suitable.
Hello Michael Essien Sakyi
The following would be a better option.
I would suggest you add cell lysis buffer to the cell pellet and carry out lysis immediately. Then you could perform a quick-freeze of the cell lysate in liquid nitrogen and store the cell lysate at - 80 degree C for future use. It would be best to freeze the lysate before clarification, since the freeze-thaw cycle may cause some denatured protein aggregates.
On the analysis day, clarify the stored cell lysate by centrifugation at 10000 to 12000 rpm at 4 degree C for 10 minutes. Use the supernatant for Western Blot.
Storing the cell pellet after centrifugation at -80 degree celsius or adding cell lysis buffer to the cell pellet and storing at -80 degree celsius is not recommended as it may degrade some protein molecules.
Best.
I think that the cell pellet should be ok without the lysis buffer if stored at -80. Alternatively, if you want them pre-lysed, you can go one step further and add your 4x and the reducing agent to your clarified lysate before freezing. This is how we always stored our samples. It's significantly more stable than storing them in straight lysis buffer and we never had issues with protein degradation even stored at -20.
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