Ultrastructural localization of nuclear matrix proteins in HeLa cells using silver-enhanced ultra-small gold probes.
We describe a method for immunogold staining of nuclear matrix proteins using ultra-small gold particles. The nuclear matrix of HeLa cells is obtained by two fractionation steps: (a) cell permeabilization with Triton X-100 to isolate the cytoskeleton, and (b) nuclease digestion followed by an incubation in 0.25 M ammonium sulfate to isolate the nuclear matrix. To prevent redistribution of internal matrix proteins during nuclear matrix preparation, pre-fixation with 0.1% acrolein was performed. Under this condition up to 80% of protein and 90% of DNA and RNA could be removed on nuclear matrix isolation, without redistribution of internal nuclear matrix proteins. For immunogold labeling, 1-nm gold probes appeared to be required to obtain optimal penetration into the nucleus. These particles can be visualized after silver enhancement. After gold labeling the matrices are stained, embedded in Epon, and ultra-thin sections are prepared for examination in the electron microscope. The applicability of this method is examplified by the localization of a 125 KD internal nuclear matrix protein and the lamins A and C in nuclear matrix preparations of HeLa cells.
de Graaf
A
A
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
van Bergen en Henegouwen
P M
PM
Meijne
A M
AM
van Driel
R
R
Verkleij
A J
AJ
D000645:Ammonium Sulfate / Q000494:pharmacology; D034961:Antigens, Nuclear; D002463:Cell Membrane Permeability / Q000187:drug effects; D003599:Cytoskeleton / Q000737:chemistry / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D034882:Lamins; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D015530:Nuclear Matrix / Q000737:chemistry* / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000032:analysis* / Q000187:drug effects / Q000302:isolation & purification; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology
D034961:Antigens, Nuclear; D034882:Lamins; D009687:Nuclear Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; D000645:Ammonium Sulfate