Enhanced nuclear proteomics.
Nuclear proteomics provides an opportunity to examine protein effectors that contribute to cellular phenotype. Both the quality and sensitivity of gel-based nuclear proteomics are limited, however, by the over-representation of histones in the protein mixture. These highly charged proteins overshadow rare species and interfere with IEF. A nuclear isolation and protein extraction procedure, tested on human embryonic stem cells, is reported that effectively isolates intact nuclei and then depletes the sample of histones by taking advantage of their ability to form an insoluble complex with DNA at lower pH (even under denaturing conditions). Ubiquitous histones and abundant nuclear actin, are depleted up to 99 +/- 0.02 and 42 +/- 5%, respectively. This technique greatly improves electrofocusing efficacy and nearly doubles the number of detected protein spots. This approach to nuclear protein isolation for 2-D PAGE opens the door to better investigation of nuclear protein dynamics.
Barthéléry
Miguel
M
Department of Pharmacology, The Pennsylvania State University College of Medicine, PA, USA.
Salli
Ugur
U
Vrana
Kent E
KE
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D018920:Coculture Techniques; D004247:DNA / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D053595:Embryonic Stem Cells / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology; D006657:Histones / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009687:Nuclear Proteins / Q000737:chemistry*; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*
D006657:Histones; D009687:Nuclear Proteins; D004247:DNA