Hello, I hope you are all doing well.
I have a question regarding my treated cells group. I plan to do western blot and qRT-PCR for my treated cells. However, to save time, I would like to separate my groups to 1 set for western blot and 1 set for qRT-PCR. I then would like to perform qRT-PCR to gather data from it but during this process, I would freeze the western blot group for when I finish with the qRT-PCR.
Can this be done? Are there any consequences or will the cell pellet begin to die? If so, for how long can I freeze the cell pellet?
As far as the cells rinsed in PBS scraped into a tube, and pelleted by centrifugation. The frozen pellet can be stored for several months at −80°C without any problem.
Your cells will die undoubtly, however, the components that you need for western blotting (protein) and qPCR (RNA) will be mantained at good conditions for both assays. Keep in mind that freeze and thaw cycles could damage those cell components.
Hi Eduardo,
Whenever I have to do both assays on my cells
1. I extract part of my cell pellet into SDS-PAGE lysis buffer right away and store the prepared protein extract at -20C until I run gels followed by Western.
2. Remaining half, I either proceed for RNA extraction right away or freeze part in Tissue Protect reagent (for RNA extraction later) and store in -80C until I proceed for RNA isolation and subsequent cDNA and qPCR.
I never have frozen pelleted cells for protein work later as I am concerned about the quality of the cells and its constituents once I thaw them.
Thanks
Madhavi
If you are aiming to do a whole-cell protein analysis, the frozen (-80C) will be fine. however, the whole intracellular architecture of the cell will be destroyed. So any type of experiments aimed at organelle-specific analysis will be completely erroneous.
Thank you for the replies. My main concern was mostly for the RNA extraction. I was going to freeze the RNA extracts at -80C but I think, based on what I learned here, that it would be best to freeze the cell pellet instead to preserve the integrity of the RNA while my primers for qRTPCR arrives.
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