Storage of whole cell lysates - How? (Jan/26/2006 )
I would like to ask a few questions:
1. Is it better to store the cell pellet or the lysate at -80C? That is, should I lyse the cells, then store at -80C, or should I store the cells at -80C, then thaw and lyse the cells when I am going to use the lysate (for SDS-PAGE and Western blotting)?
I've asked a few people. They say that the cell pellet is generally better since you are freezing things in tact.
2. Is it better to snap freeze in liquid nitrogen, then place at -80C? Or better to just stick it at -80C without snap freezing?
3. How do you harvest cells at different time points? Say I am harvesting at 0, 1hr, 5hr, and 12hr. Will you spin down the cells and store the cell pellet at -80C until you have the 12hr time point, at which point you would thaw all the cells and lyse them at once?
Or will you lyse the cells at each time point and store at -80C? Thanks
hi
i prefer store cell lysates rather than whole cells as protease/nucleases may have effect when thawing. i do that with no pb for many years.
For freezing, i just put the eppis in box and all at -80°. But for transcription assay, a colleague noticed that better activity is obtained by nitrogen freezing.
1. Is it better to store the cell pellet or the lysate at -80C? That is, should I lyse the cells, then store at -80C, or should I store the cells at -80C, then thaw and lyse the cells when I am going to use the lysate (for SDS-PAGE and Western blotting)?
I've asked a few people. They say that the cell pellet is generally better since you are freezing things in tact.
2. Is it better to snap freeze in liquid nitrogen, then place at -80C? Or better to just stick it at -80C without snap freezing?
3. How do you harvest cells at different time points? Say I am harvesting at 0, 1hr, 5hr, and 12hr. Will you spin down the cells and store the cell pellet at -80C until you have the 12hr time point, at which point you would thaw all the cells and lyse them at once?
Or will you lyse the cells at each time point and store at -80C? Thanks
Hey,
1. I usually snap freeze my cells in liquid nitrogen and then store them at -80 until I am ready to lyse them. In fact, I have done that without even lifting the cells from the plate (wash plates 3X with cold PBS (or other) first and then freeze plate in liquid nitrogen). In theory, freezing them may help with lysis. Once lysed I usually don't btoher with flash freezing and just dump them at -80.
2. I would think it would be preferable to flash freeze than just placing at -80 (stop everything in their tracks but that is not necessarily true depends on what you are lysing with.
3. Depends if you are going to use them soon after your 12hr time point or if you are going to leave them for another time. If using them right away I would make my lysates after each time point (only if there was enough time between time points). If wait, I would freeze them.
Take care and Good luck