I'm culturing Caco2 cells on a two 6-well plate inserts, one for Rna experiment and the other one for western analysis. Knowing that I want in the first place to collect my samples and store them at -80°C for later use.
1) Ist good to collect the Caco2 from the inserts by adding (500-800µl) Trizol and store them at this level at -80°C? or shall I centrifuge, aspirate the supernatant and store only the cell pellet at -80°C? (Regarding RNA plate)
2) For the Western plate, Can I resuspend the harvested cell pellet in Laemmli buffer and store it at this level at -80°C for later use? or it's better to store the cell pellet in PBS at -80°C , then for later I start the experiment by thawing the pellet in laemmli or any other lysis buffer?
Thank you!
Hello Jamila Hijazi
1. For the RNA plate:
You should collect the Caco2 cells from the inserts by adding (500-800µl) TRIzol and store them at this level at -80°C. Please note that TRIzol is a monophasic solution of phenol and guanidinium isothiocyanate that simultaneously solubilizes biological material and denatures protein. After solubilization, the addition of chloroform causes phase separation (much like extraction with phenol:chloroform:isoamyl alcohol), where protein is extracted to the organic phase, DNA resolves at the interface, and RNA remains in the aqueous phase. Therefore, RNA, DNA, and protein can be purified from a single sample.
Please do not follow the alternative method that you have mentioned namely, “shall I centrifuge, aspirate the supernatant and store only the cell pellet at -80°C”.
2. For the Western plate:
I would suggest that you harvest the cells and carry out cell lysis in ice-cold lysis buffer (preferably RIPA lysis buffer containing protease inhibitors). Then you may store your sample after lysis in RIPA buffer at -80 degree C in aliquots such that each aliquot is just enough for the number of gels you would have to run at a time. This will help to avoid freeze-thaw cycles as repeated freeze-thaw would cause degradation of your protein of interest.
Do not store the cell pellet at -80 degree C. Instead, carry out the cell lysis as mentioned above and store the cell lysate which is in RIPA buffer containing protease inhibitors at -80 degree C for later use.
Good Luck!
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