Efficient sequential recovery of nucleolar macromolecular components.
Efficient extraction and accurate quantification of nucleolar macromolecules are critical for in vitro analysis, especially for studying RNA, DNA, and protein dynamics under identical conditions. There is presently no single method that efficiently and simultaneously isolates these three macromolecular constituents from purified nucleoli. We have developed an optimized method, which without evident loss, extracts, and solubilizes protein recovered from a single sample following TRIzol isolation of RNA and DNA. The solubilized protein can be accurately quantified by protein bicinchoninic acid assay and assessed by polyacrylamide gel electrophoresis. We have successfully applied this approach to extract and quantify all three nucleolar components, and to study nucleolar protein responses after actinomycin D treatment.
Sidney Kimmel Comprehensive Cancer Center, Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
Baoyan
B
Bai
Marikki
M
Laiho
D002466:Cell Nucleolus / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D006146:Guanidines / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D010636:Phenols / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D011804:Quinolines / Q000737:chemistry; D015138:RNA, Nuclear / Q000737:chemistry / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000737:chemistry
D006146:Guanidines; D009687:Nuclear Proteins; D010636:Phenols; D011804:Quinolines; D015138:RNA, Nuclear; C411644:trizol; D004247:DNA; C047117:bicinchoninic acid