Hi good people,
I have got frozen vials of cell lines. I need to know how should I start with this vial for protein extraction? The thawing temperature, the centrifugation speed and all??
Kindly help.
Sumera
Do you mean to passage the cells?
If you are going to passage the cells just thaw them in Luke warm water for 1-2 minutes, wash the cells to get rid of freezing medium by adding cell culture medium and spinning them down 1500 rcf for 5 minutes and put them in cell culture flask with media having serum in it. I hope so it helps
No.I am not talking about passaging.
My question is how to prepared frozen cells for protein extraction.
What Charles said. In your case I would first try thawing the frozen cells on ice (it will take a long time but since you don't care about long term cell viability it shouldn't be a problem) wash (add and then centrifuge 0C-4C 10 minutes 500-1000 rpm) the cells with ice cold serum free cell culture media once (maybe let them sit in this for a bit after first centrifugation) then at least 3 times with ice-cold PBS to remove the dead cells, freezing media, and regular media components. For extra protection against degradation I recommend the Cell signaling technology lysis buffer with roche complete protease and phosphatase inhibitors for your cell lysis buffer cocktail.
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