I have PBMC samples. My protein of interest is a mitochondrial. So I need to lyse the cells. Mitochondrial protein isolation seems to be a good option, but ELISA method by the manufacture have suggested cell lysis technique. Can anyone suggest me the proper freeze thaw technique?
Just give your mct's containing cell suspension a quick dip into liquid nitrogen and thaw them.
There is an interesting resource on traditional methods for cell lysis on the ThermoFisher website where you will find explanation for your freeze-thaw technique. Only thing I would add is to check after every freeze-thaw cycle the efficiency of your procedure by trypan blue staining. Also you may want to consider using a dounce homogenizer for cell lysis. It is a quite fast and efficient method and it is generally recommended for protocols involving mitochondria isolation.
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