ORIGINAL PAPER Nuclear protein extraction from frozen porcine myocardium Diederik W. D. Kuster & Daphne Merkus & Huub J. J. Jorna & Dick H. W. Dekkers & Dirk J. Duncker & Adrie J. M. Verhoeven Received: 19 July 2010 / Accepted: 15 October 2010 / Published online: 9 November 2010 # University of Navarra 2010 Abstract Protocols for the extraction of nuclear proteins have been developed for cultured cells and fresh tissue, but sometimes only frozen tissue is available. We have optimized the homogenization procedure and subsequent fractionation protocol for the preparation of nuclear protein extracts from frozen porcine left ventricular (LV) tissue. This method gave a highly reproducible protein yield (6.5±0.7% of total protein; mean±SE, n =9) and a 6-fold enrichment of the nuclear marker protein B23. The nuclear protein extracts were essentially devoid of cytosolic, myofil- ament, and histone proteins. Compared to nuclear extracts from fresh LV tissue, some loss of nuclear proteins to the cytosolic fraction was observed. Using this method, we studied the distribution of tyrosine- phosphorylated signal transducer and activator of transcription 3 (PY-STAT3) in LV tissue of animals treated with the β-agonist dobutamine. Upon treat- ment, PY-STAT3 increased 30.2±8.5-fold in total homogenates, but only 6.9±2.1-fold (n =4, P=0.03) in nuclear protein extracts. Of all PY-STAT3 formed, only a minor fraction appeared in the nuclear fraction. This simple and reproducible protocol yielded nuclear protein extracts that were highly enriched in nuclear proteins with almost complete removal of cytosolic and myofilament proteins. This nuclear protein extraction protocol is therefore well-suited for nuclear proteome analysis of frozen heart tissue collected in biobanks. Keywords Nuclear extract . Frozen tissue . Transcription factors . Heart tissue Abbreviations DPBS Dulbecco’ s phosphate-buffered saline ECL Enhanced chemiluminescence GAPDH Glyceraldehyde-3-phosphate dehydrogenase LV Left ventricle LV dP/dt max Maximum rate of rise of left ventricular pressure PY-STAT3 Tyrosine-phosphorylated signal transducer and activator of transcription 3 SERCA2 Sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 2 SR Sarcoplasmic reticulum TF Transcription factor J Physiol Biochem (2011) 67:165–173 DOI 10.1007/s13105-010-0059-x D. W. D. Kuster : H. J. J. Jorna : D. H. W. Dekkers : A. J. M. Verhoeven (*) Department of Biochemistry, Cardiovascular Research Institute COEUR, Erasmus University Medical Center, PO Box 2040, 3000 CA Rotterdam, The Netherlands e-mail: a.verhoeven@erasmusmc.nl D. W. D. Kuster : D. Merkus : D. J. Duncker Division of Experimental Cardiology, Department of Cardiology, Thoraxcenter, Cardiovascular Research Institute COEUR, Erasmus University Medical Center, Rotterdam, The Netherlands