Premium
Simultaneous extraction of nucleic acids and proteins from tissue specimens by ultracentrifugation: A protocol using the high‐salt protein fraction for quantitative proteome analysis
Author(s) -
Grzendowski Michael,
Riemenschneider Markus J.,
Hawranke Eva,
Stefanski Anja,
Meyer Helmut E.,
Reifenberger Guido,
Stühler Kai
Publication year - 2009
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200800902
Subject(s) - nucleic acid , ultracentrifuge , homogenization (climate) , proteome , chemistry , dna , chromatography , rna , analytical ultracentrifugation , biochemistry , microbiology and biotechnology , biology , gene , biodiversity , ecology
Comprehensive molecular profiling of human tumor tissue specimens at the DNA, mRNA and protein level is often obstructed by a limited amount of available material. Homogenization of frozen tissue samples in guanidine isothiocyanate followed by ultracentrifugation over cesium chloride allows the simultaneous extraction of high‐molecular weight DNA and RNA. Here, we present a protocol for quantitative proteome analysis using the high‐salt protein fraction obtained as supernatant after ultracentrifugation for nucleic acid extraction. We applied this method to extracts from primary human brain tumors and demonstrate its successful application for protein expression profiling in these tumors using 2‐D DIGE, MS and Western blotting.