Subfractionation of heart tissue: the "in sequence" myofilament protein extraction of myocardial tissue.
Proteomic analysis of heart tissue is complicated by the large dynamic range of its proteins. The most abundant proteins are the myofilament proteins, which comprise the contractile apparatus. This chapter describes a protocol for fractionation of heart tissue that extracts the myofilament proteins into a separate sample fraction, allowing analysis of lower-abundance proteins. Importantly, this is performed in a manner that is compatible with two-dimensional electrophoresis and high-performance liquid chromatography, two of main technologies of proteomics. The method produces three fractions based on solubility at different pHs: (1) cytoplasmic-enriched extract (neutral pH), (2) myofilament-enriched extract (acidic pH), and (3) membrane protein-enriched pellet. Fractionation of heart tissue in this manner provides the basis for in-depth proteomic analysis.
Kane
Lesley A
LA
Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD, USA.
Neverova
Irina
I
Van Eyk
Jennifer E
JE
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D006801:Humans; D008840:Microfilament Proteins / Q000032:analysis*; D009206:Myocardium / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism
D008840:Microfilament Proteins