how to extract nuclear and cytoplasmic protein from trizole after RNA extraction?
i want to extract nuclear and cytoplasmic faction from sample after RNA extraction using chloroform, iso-propanol and EtOH, i have the procedure of protein extraction after RNA extraction using Trizole however during this extraction the protein is left as an pellet. using this procedure is there any protocol that allow me to extract the nuclear and cytoplasmic fraction
(ps. the pellet is then dilute in water and sonificate on ice to broke the pellet)
I (and many others in our lab) have never had any luck even resuspending the protein pellet from a Trizol extraction. The protein pellet after a Trizol extraction is also all the protein from all fractions of the cells (i.e. nuclear, cytoplasmic, mitochondrial, etc.). This is because all the organelles are lysed with the Trizol reagent. Because of that, you unfortunately won't be able to extract separate nuclear and cytoplasmic fractions from your Trizol protein pellet.
There are protocols to fractionate samples, and our lab uses one for brain samples to fractionate mitochondrial, nuclear, and cytoplasmic fractions. We use this to assess things like translocation between compartments (e.g. cytoplasmic to nuclear translocation).
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