Nuclear protein extraction from frozen porcine myocardium.
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, myofilament, 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 treatment, 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.
Department of Biochemistry, Cardiovascular Research Institute COEUR, Erasmus University Medical Center, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
Diederik W D
DW
Kuster
Daphne
D
Merkus
Huub J J
HJ
Jorna
Dick H W
DH
Dekkers
Dirk J
DJ
Duncker
Adrie J M
AJ
Verhoeven
D000818:Animals; D009206:Myocardium / Q000378:metabolism*; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis*; D050796:STAT3 Transcription Factor / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine; D014443:Tyrosine / Q000378:metabolism
D009687:Nuclear Proteins; D020543:Proteome; D050796:STAT3 Transcription Factor; D014443:Tyrosine