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Nuclear protein extraction from frozen porcine myocardium

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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, 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.

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Abbreviations

DPBS:

Dulbecco’s phosphate-buffered saline

ECL:

Enhanced chemiluminescence

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

LV:

Left ventricle

LV dP/dtmax :

Maximum rate of rise of left ventricular pressure

PY-STAT3:

Tyrosine-phosphorylated signal transducer and activator of transcription 3

SERCA2:

Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2

SR:

Sarcoplasmic reticulum

TF:

Transcription factor

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Acknowledgment

This work was supported by a grant from the Netherlands Heart Foundation (NHS2005B234).

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Correspondence to Adrie J. M. Verhoeven.

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Kuster, D.W.D., Merkus, D., Jorna, H.J.J. et al. Nuclear protein extraction from frozen porcine myocardium. J Physiol Biochem 67, 165–173 (2011). https://doi.org/10.1007/s13105-010-0059-x

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