cytoplasmic and nuclear extraction - (May/09/2010 )

Hi all,

I have searched the archives of this forum and yet I will throw this question again just because I am still confused of the final conclusion of the answer to the question.

I am trying to separate the nuclear and cytoplasmic protein from my cultured cell lines (two of them), and am using the NE-PER kit. I followed the protocol to separate them and then ran western on them and confirmed by a dot blot that there are more expression of my transcription factor that is supposedly in the cytoplasm in rested cells. At the same time, there are more actin in nuclear fraction than in cytoplasm. I am concerned because a colleague of mine used this method on this exact same cell line and told me that I am supposed to get no actin in my nuclear fraction if I separate them well. So the possibility is that I did not get well enough of a fractionation. Even then, I am confused of the use of actin as 'fraction control'. Some people in this forum have claimed that actin is not ideal to be used as a mean to see if I get clean separation, and other protein such as histone, lamin or nucleolin, p53 might be better for nucleus, and tubulin for cytoplasm. I then called the NE-PER guyz and they confirmed that actin may not be a good marker, since they are also found in nucleus. I then wonder of why my colleague would see no actin in the nuclear fraction, and whether this means that actin can be used as fraction control?

Then another question is, is there another way to ensure that the nuclear or cytplasm is properly fractionated other than using western with those markers?

Actin is found in the cytoskeleton of the cell, it is a component of both the nuclear membrane and the cytoplasm, therefore it can not be used as a control for fractionation. I don't know of any way of determining whether you have good fractionation other than running westerns for nuclear and cytoplasmic proteins. I guess you could look for the presence of DNA in the nuclear fraction, but most DNA detection methods will also pick up RNA which will be found in both nucleus and cytoplasm.

We use Histone and Tubulin as controls for our fractionations.

nuclelolin shuttles proteins from cytoplasm to nucleus and can also be found on plasma membrane. it is not a good marker for nuclear extraction. i've had better luck with histone.