As I am going on with my proteomic approach. Now, I am stuck at one step I want to look inside the nucleus protein expression profile of different tissues. I am finding some of the kits available with same aim but they are not good for nucleus membrane and chromatin binding proteins and I want to cover all proteins with special emphasis of Chromatin binding proteins. So, I am looking for an nucleus isolation protocol form normal and cancerous tissue or an effective protocol that can avail me chromatin associated proteins.
If any one working with same approach or same kind of work or having experience with same kind of work then kindly share your knowledge and expertise.
Looking for other Idea and suggestions.
I would like to serve any further information if required.
Hi Amit,
most people would use a variation of Dignam et al. 1983, NAR. Here is one example:
http://www.lifesci.dundee.ac.uk/groups/sonia_rocha/Protocols/Nuclear%20extracts%20protocol_SRLab.pdf
What you want are the nuclei (pellet after first spin) before bursting them with hypothonic buffer.
Nevertheless enrichment by subcellular fractionation might not give you that much of an enrichment. You would still find a lot of proteins from other organelles and the cytoplasm. You can try to perform the analyses with and without subcellular fractionation and see if you have enough coverage. Modern mass spectrometry based proteomics can give you quite a good coverage without fractionation.
Here is one example how to evaluate the organellar enrichment "degree":
http://www.mcponline.org/content/7/4/672.full (fig. 2)
1. add TE buffer containing proteinase K (200ul) final vol. to 5 mg tissue , incubate overnight at 50C.
vortex gently , add 2% SDS (50ul) mix and incubated at 50C for 15min. add equal vol. chloroform and mix gently , centrifuge for 10min 12xg , separate the superannuate that containing total nucleic acid
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