A rapid method is described for the isolation of nuclei in high yield from L8E63 rat myoblast and myotube cells in culture. Cells are lysed in hypotonic buffer (pH 7.8) containing 3 mM CaCl21 mM EDTA, and protease inhibitors, Dounce homogenized, and washed to obtain purified nuclei. Based on DNA recovery, the isolated fraction contains greater than 80% of the nuclei in the original homogenate. Phase- and electron-microscopic examinations reveal that the purified nuclei are well preserved, have intact double membranes, and are morphologically similar to nuclei observed in situ. Furthermore, the cytoplasmic contamination as fragments of cytomembrane and fibrillar components is minimal. The protein: DNA and RNA: DNA ratios are similar to most mammalian nuclei. We have fractionated developmental stage-specific chromatin from purified nuclei obtained from proliferating and fused L8E63 muscle cells, as detailed previously . Biochemical and spectral analyses of the chromatin preparations show protein: DNA ratios similar to that found in other mammalian tissues and cells, but the RNA content is lower. Contamination of the chromatin protein by the cytoplasmic protein and membrane phospholipid (as choline) is acceptably low. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) reveals some quantitative and qualitative stage-specific differences in the distribution of the non-histone chromosomal proteins. Two of the major non-histone proteins in myotube chromatin have molecular weights (MW) similar to muscle myosin and actin. Their presence in myotube chromatin is discussed.
|Number of pages||14|
|Journal||Experimental Cell Research|
|State||Published - Jan 1982|
Bibliographical noteFunding Information:
We thank Jon Charlesworth of this departmentf or doing the electronm icroscopy.This researchw as supported in part by grants from the Graduate Research Board, University of Illinois, and the NIH Grant No. GM 27236. I. M. is a pre-doctoral fellow of the Illinois Institutef or DevelopmentalD isabilities.
ASJC Scopus subject areas
- Cell Biology