Isolation and characterization of the nuclear proteome

A method for isolating samples enriched in nucleoplasm associated transcription factors, euchromatin associated transcription factors, and heterochromatin associated transcription factors from cells, the method comprising:

• (a) suspending cells in a first buffer that does not comprise polycations;
• (b) adding a detergent solution to the suspended cells;
• (c) separating nuclei from the cells following (b);
• and
• sequentially extracting the nucleoplasm associated transcription factors, euchromatin associated transcription factors, and heterochromatin associated transcription factors from the separated nuclei as follows:
• (d) re-suspending the nuclei of (c) in a second buffer not comprising polycations but comprising ethylenediaminetetraacetic acid (EDTA) and salt;
• (e) separating insoluble chromatin after (d);
• (f) collecting a first supernatant liquid after (e), wherein the first supernatant liquid is enriched for nucleoplasm associated transcription factors;
• (g) re-suspending the insoluble chromatin of (e) in a third buffer not comprising polycations but comprising EDTA and an increased salt concentration as compared to the second buffer to extract euchromatin associated transcription factors;
• (h) separating insoluble chromatin after (g);
• (i) collecting a second supernatant liquid after (g), wherein the second supernatant liquid is enriched for euchromatin associated transcription factors;
• (j) re-suspending the insoluble chromatin of (h) in a fourth buffer not comprising polycations but comprising EDTA and an increased salt concentration as compared to the third buffer to extract heterochromatin associated transcription factors;
• (k) separating insoluble chromatin after (j);
• and
• (l) collecting a third supernatant liquid after (k), wherein the third supernatant liquid is enriched for heterochromatin associated transcription factors,
• wherein the method further comprises analyzing and identifying the nucleoplasm associated transcription factors in the first supernatant liquid, the euchromatin associated transcription factors in the second supernatant liquid, and/or the heterochromatin associated transcription factors in the third supernatant liquid by mass spectrometry, and
• wherein histone proteins remain in the insoluble chromatin of (k).