Linked Life Data

19081104

Source:http://linkedlifedata.com/resource/pubmed/id/19081104

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
17
pubmed:dateCreated
2009-4-6
pubmed:abstractText
Several methods have been described in the literature for removal of DNA from protein samples prior to proteome analysis. They in general involve protein precipitation techniques. In other protocols, DNAse treatment is suggested prior to precipitation of proteins in excess acetone. All these methods have been evaluated and found to perform poorly in DNA removal, as illustrated by two-dimensional (2D) maps where horizontal and vertical sample streaking are still substantial. Such removal is in general necessary in tissue lysates and especially when analysing sub-cellular organelles, such as nuclei, where the high DNA levels strongly interfere with proteome analysis. Another method is proposed here for efficient DNA removal: two-phase extraction of DNA in chloroform/phenol/isoamyl alcohol, a procedure commonly used to rid DNA samples of protein contaminants, but rarely applied to protein preparation. This extraction is not very efficient if performed at slightly acidic to neutral pH values, but it performs extremely well at pH values of 9.5 or higher. The 2D maps thus obtained of Escherichia coli lysates as well as extracts from purified nuclei of eukaryotic cells are not only devoid of any vertical or horizontal streaking, but exhibit many more spots, especially in the alkaline region of the 2D gels, suggesting that these basic proteins were in general lost to proteome analysis due to co-precipitation in tenacious protein-DNA complexes. It is hypothesized that the alkaline pH values adopted in the two-phase extraction help to fully disrupt any residual DNA-protein complexes, due to strong Coulombic repulsion.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1873-3778
pubmed:author
pubmed:issnType
Electronic
pubmed:day
24
pubmed:volume
1216
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
3606-12
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed-meshheading:19081104-Bacterial Proteins, pubmed-meshheading:19081104-Cell Line, Tumor, pubmed-meshheading:19081104-Cell Nucleus, pubmed-meshheading:19081104-Chemical Fractionation, pubmed-meshheading:19081104-Chemical Precipitation, pubmed-meshheading:19081104-Chloroform, pubmed-meshheading:19081104-DNA, pubmed-meshheading:19081104-Electrophoresis, Gel, Two-Dimensional, pubmed-meshheading:19081104-Endodeoxyribonucleases, pubmed-meshheading:19081104-Endoribonucleases, pubmed-meshheading:19081104-Escherichia coli, pubmed-meshheading:19081104-Humans, pubmed-meshheading:19081104-Hydrogen-Ion Concentration, pubmed-meshheading:19081104-Mass Spectrometry, pubmed-meshheading:19081104-Pentanols, pubmed-meshheading:19081104-Phenol, pubmed-meshheading:19081104-Proteins, pubmed-meshheading:19081104-Proteomics, pubmed-meshheading:19081104-Reproducibility of Results
pubmed:year
2009
pubmed:articleTitle
Efficient removal of DNA from proteomic samples prior to two-dimensional map analysis.
pubmed:affiliation
Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, Milan, Italy.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't, Evaluation Studies