I've been using commercial kits to separate cytoplasmic and nuclear proteins. I noticed that many advise against using their kits for frozen tissue because the nuclear membrane can break and leak into the cytoplasmic portion. However, all my tissue have to be frozen because I bring them from another facility. Has anyone come across a good method to separate cytoplasmic and nuclear proteins from frozen tissues or account for the slight leakage? They have only been frozen and thawed once before analysis via western blot.
Thank you -- Greg
Given that your available sample is quite small in amount but stored in fresh conditions, I strongly recommend you to use the NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit to obtain both the cytoplasmic fraction protein and cytoplasmic fraction. (strictly speaking, which includes cellular membrane proteins) from thawed tissue.
https://www.fishersci.com/shop/products/thermo-scientific-ne-per-nuclear-cytoplasmic-extraction-kit/p-2712560
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