Nuclear protein in a post nuclear supernatant? - (Feb/08/2006 )

Hi, I have to transfect a plasmid encoding a protein which contains a nuclear localitzation signalling peptide in his sequence. Will I be able to detect the protein by Western blot in a postnuclear supernatant from the lysate of the transfected cells?

Thanks in advance.

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hi
hat do you mean by post nuclear supernatant ?

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I mean with postnuclear supernatant or PNS, after scrapping the cells from the plate (with lysis buffer with detergent) make a centrifugation at 18000g during 15', to pull down the mebranes. The supernatant of this centrifugation is called PNS (which in principle I think it shouldn't contain nuclear proteins, that's my doubt).

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hi
i don't knw this protocol for nuclear proteins extract...
you can try to detect p53 which should be nuclear.
Best thing is to do a westrn on this supernatant and with proteins extracted from the pellet.

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why would you not do the usual extraction, with a cytoplasmic and a nuclear fraction, and then do a western to make sure the protein is going to the nucleus where it belongs?

I suppose I am not sure what you are asking?

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Yes, I could possibly explain better the problem. My doubt actually is the next: if I transfect a plasmid which encodes a protein with a nuclear localitzation peptide in his sequence, I am supposed to find the protein in the nuclear fraction, am not I? The question can look obvious but it has a reason: we detect it in the cytosol fraction by western blot ¿?

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You may want to immunolocalize the protein.
This way you will have an independent method to:
-check that your protein is actually targeted to the nucleus
-asses that your nuclear/cytoplasmic fractionation protocol is good or sucks.
Good luck

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