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Enhanced nuclear proteomics
Author(s) -
Barthéléry Miguel,
Salli Ugur,
Vrana Kent E.
Publication year - 2008
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200700841
Subject(s) - proteomics , histone , nuclear protein , quantitative proteomics , biology , isoelectric focusing , isoelectric point , protein purification , biochemistry , chemistry , microbiology and biotechnology , dna , enzyme , gene , transcription factor
Nuclear proteomics provides an opportunity to examine protein effectors that contribute to cellular phenotype. Both the quality and sensitivity of gel‐based nuclear proteomics are limited, however, by the over‐representation of histones in the protein mixture. These highly charged proteins overshadow rare species and interfere with IEF. A nuclear isolation and protein extraction procedure, tested on human embryonic stem cells, is reported that effectively isolates intact nuclei and then depletes the sample of histones by taking advantage of their ability to form an insoluble complex with DNA at lower pH (even under denaturing conditions). Ubiquitous histones and abundant nuclear actin, are depleted up to 99 ± 0.02 and 42 ± 5%, respectively. This technique greatly improves electrofocusing efficacy and nearly doubles the number of detected protein spots. This approach to nuclear protein isolation for 2‐D PAGE opens the door to better investigation of nuclear protein dynamics.