US 12,276,667 B2
Isolation and characterization of the nuclear proteome
Alexander Federation, Seattle, WA (US); and John Stamatoyannopoulos, Seattle, WA (US)
Assigned to Altius Institute for Biomedical Sciences, Seattle, WA (US)
Filed by Altius Institute for Biomedical Sciences, Seattle, WA (US)
Filed on May 21, 2019, as Appl. No. 16/417,658.
Claims priority of provisional application 62/646,298, filed on Mar. 21, 2018.
Prior Publication US 2020/0033358 A1, Jan. 30, 2020
Int. Cl. G01N 33/68 (2006.01); C12N 9/16 (2006.01)
CPC G01N 33/6848 (2013.01) [C12N 9/16 (2013.01); C12Y 301/31001 (2013.01)] 15 Claims
OG exemplary drawing
 
1. 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).