Differential silver-staining sodium dodecyl sulfate-polyacrylamide gel electrophoresis: a nonisotopic method for characterizing gel-separated histone-DNA complexes.
Some nonspecific, DNA-binding proteins, like the linker histones, precipitate DNA upon binding. This is a poorly understood process that limits analysis of such nucleoprotein complexes using standard gel electrophoresis. To circumvent this problem, low concentrations of glutaraldehyde were used to crosslink the linker histones to DNA; then the partially crosslinked complexes were solubilized in SDS2 and separated by SDS-PAGE. Differential detection was accomplished using two different silver staining protocols that preferentially stained either proteins or nucleic acids. A technique was developed which allows the relative proportion of linker histones and DNAs in each detected band to be determined, and is referred to as differential staining SDS-PAGE (DS-SDS-PAGE). DS-SDS-PAGE provides a novel, non-isotopic means for characterizing multiple nucleoprotein bands separated by polyacrylamide gel electrophoresis. In applying this method to a model linker histone-DNA study, we were able to detect both protein-DNA and protein-protein contacts that are important in linker histone assembly onto DNA.
Carter
G J
GJ
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305, USA.
van Holde
K
K
D000818:Animals; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D003959:Diamines; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005976:Glutaral / Q000737:chemistry; D006657:Histones / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D016622:Silver Staining / Q000379:methods*; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin / Q000378:metabolism
D003432:Cross-Linking Reagents; D003959:Diamines; D006657:Histones; D012967:Sodium Dodecyl Sulfate; D004247:DNA; D014357:Trypsin; D005976:Glutaral