Could anyone recommend the most appropriate housekeeper for nuclear and cytosolic fraction? I want to isolate both fractions from frozen rat brain tissues and am wondering what is the best and most commonly used housekeeper to quantify changes in another protein of interest using Western blot?
TBP is for nuclear, and cyclophlin A is for cyto. What I did is cell culture, and I think they should also work well with tissues. Good luck.
I 'would be afraid, the freezing already destroyed the organelles in your tissues, mitochondria definitely. Try the fractionation on the new brain (fresh/and frozen), before you will destroy your samples by defreezing. I'm sorry to say it, but you can have the situation your protein is moving because of the freezing process even when you observe fine nuclear fraction.
I agree with the suggestions posted here, but I would add GAPDH or Na+/K+ exchanger for cytosolic fractions and Histone H3 or Lamins for nuclear. Just to increase the chances for you to find a suitable antibody!
Andre, I cannot use GAPDH as a cytosolic marker as I found changes in GAPDH gene expression in hippocampus following stress exposure.
Well, anybody working with brain tissues? It seems there are plenty of markers of course, but that is the point, to find the most suitable for frozen brain tissues.
So, which one you would consider " a winner" ? :-)
Hi Jonathan, yes, I already did it and published successfully, though it depends whether you are using frozen or fresh tissue. With frozen one do not expect 100% purity since the cells are already disturbed. I used snap frozen brain tissue and got nice purity though nuclear fraction was little bit contaminated by cytosolic one. Please, see attached paper with the method I used and let me know how it worked for you. Also there is a kit from Qiagen or other company for fractionation, but I have not tried it. It also suggest to use fresh tissue if possible. Good luck.
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