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Nuclear protein extraction from frozen porcine myocardium
Author(s) -
Kuster Diederik,
Merkus Daphne,
Dekkers Dick,
Duncker Dirk Jan,
Verhoeven Adrie
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb246
Subject(s) - nuclear protein , histone , homogenization (climate) , chemistry , cell nucleus , nuclear transport , microbiology and biotechnology , biology , biochemistry , dna , gene , transcription factor , biodiversity , ecology
Extraction of nuclear proteins from cells or tissues is an important tool for studying gene regulation and the responsible transcription factors. Here we present a homogenization and fractionation protocol for the preparation of nuclear extracts from frozen porcine left ventricular (LV) tissue. This nuclear extraction procedure gave a highly reproducible protein yield (7.3 ± 0.3% of total protein; mean ± SE, n= 11), a six‐fold enrichment of the nuclear marker B23, and more than 40% recovery of B23. The nuclear extracts were essentially devoid of contamination with cytosolic, myofilament and histone proteins as shown by Western blotting. Using this nuclear extraction method we show that upon treatment of animals with the β‐adrenergic agonist dobutamine, the amount of tyrosine phosphorylated STAT3 increased 6.9 ± 2.1 fold (n = 4, P = 0.03) in nuclear extracts from LV tissue, but only a minor fraction of the total PY‐STAT3 appeared in the nucleus. This nuclear extraction method appears to be well‐suited to nuclear proteome analysis of frozen heart tissue collected in biobanks. This work was supported by a grant from the Netherlands Heart Foundation (NHS2005B234).