Simultaneous extraction of nucleic acids and proteins from tissue specimens by ultracentrifugation: A protocol using the high-salt protein fraction for quantitative proteome analysis.
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.
Grzendowski
Michael
M
Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
Riemenschneider
Markus J
MJ
Hawranke
Eva
E
Stefanski
Anja
A
Meyer
Helmut E
HE
Reifenberger
Guido
G
Stühler
Kai
K
D001932:Brain Neoplasms / Q000737:chemistry*; D005910:Glioma / Q000737:chemistry*; D006146:Guanidines / Q000737:chemistry; D006801:Humans; D017879:Isothiocyanates / Q000737:chemistry; D009696:Nucleic Acids / Q000302:isolation & purification*; D011506:Proteins / Q000302:isolation & purification*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D015203:Reproducibility of Results; D014461:Ultracentrifugation / Q000379:methods*
D006146:Guanidines; D017879:Isothiocyanates; D009696:Nucleic Acids; D011506:Proteins; D020543:Proteome; C054435:guanidine isothiocyanate